Smart Pro-Drugs of Serine Protease Inhibitors

ABSTRACT

The present invention relates to prodrugs of protease inhibitors, such as inhibitors of the proteosome, DPP IV, FAPα and the like. These “pro-inhibitors” are activated, i.e., cleaved, by an “activated protease” to release an active inhibitor moiety in proximity to a “target protease”. The identity of activating protease and target protease can be the same (such as pro-inhibitors being referred to as “Target-Activated Smart Protease Inhibitors” or “TASPI”) or different (e.g., “Target-Directed Smart Protease Inhibitors” or “TDSPI”). After activation of the pro-inhibitor, the active inhibitor moiety can self-inactivate by, e.g., intramolecular-cyclization or cis-trans isomerization.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/512,213, filed Aug. 29, 2005; which is a national stage filing under35 U.S.C. 371 of International Application PCT/US03/013561, filed Apr.30, 2003, which designated the United States and which claims thebenefit of priority to U.S. Provisional Application No. 60/376,636,filed Apr. 30, 2002. The entire teachings of the referenced Applicationsare incorporated herein by reference. International ApplicationPCT/US03/013561 was published under PCT Article 21(2) in English.

BACKGROUND OF THE INVENTION

Proteases are enzymes that cleave proteins at single, specific peptidebonds. Proteases can be classified into four generic classes: serine,thiol or cysteinyl, acid or aspartyl, and metalloproteases (Cuypers etal., J. Biol. Chem. 257:7086 (1982)). Proteases are essential to avariety of biological activities, such as digestion, formation anddissolution of blood clots, reproduction and the immune reaction toforeign cells and organisms. Aberrant proteolysis is associated with anumber of disease states in man and other mammals. In many instances, itis beneficial to disrupt the function of one or more proteolytic enzymesin the course of therapeutically treating an animal.

The binding site for a peptide substrate consists of a series of“specificity subsites” across the surface of the enzyme. The term“specificity subsite” refers to a pocket or other site on the enzymecapable of interacting with a portion of a substrate for the enzyme. Indiscussing the interactions of peptides with proteases, e.g., serine andcysteine proteinases and the like, the present application utilizes thenomenclature of Schechter and Berger [(1967) Biochem. Biophys. Res.Commun 27:157-162)]. The individual amino acid residues of a substrateor inhibitor are designated P1, P2, etc. and the corresponding subsitesof the enzyme are designated S1, S2, etc, starting with the carboxyterminal residue produced in the cleavage reaction. The scissile bond ofthe substrate is amide bond between S1-S1′ of the substrate. Thus, forthe peptide Xaa1-Xaa2-Xaa3-Xaa4 which is cleaved between the Xaa3 andXaa4 residues, the Xaa3 residue is referred to as the P1 residue andbinds to the 51 subsite of the enzyme, Xaa2 is referred to as the P2residue and binds to the S2 subsite, and so forth.

Dipeptidyl peptidase IV (DPIV), for example, is a serine protease whichcleaves N-terminal dipeptides from a peptide chain containing,preferably, a proline residue in the penultimate position, e.g., in theP1 position. DPIV belongs to a group of cell-membrane-associatedpeptidases and, like the majority of cell-surface peptidases, is a typeII integral membrane protein, being anchored to the plasma membrane byits signal sequence. DPIV is found in a variety of differentiatedmammalian epithelia, endothelia and hemapoetic cells and tissues,including those of lymphoid origin where it is found specifically on thesurface of CD4⁺ T cells. DPIV has been identified as the leukocytedifferentiation marker CD26.

Proteosomes are serine proteases responsible for the majority ofintracellular protein turnover in eukaryotic cells, includingproteolytic degradation of damaged, oxidized or misfolded proteins, aswell as processing or degradation of key regulatory proteins requiredfor various cellular functions, such as, e.g., cell cycle progression.For example, the 26S proteosome is a multi-catalytic protease comprisingat its catalytic core the 20s proteosome, a multi-subunit complex ofapproximately 700 kDa molecular weight. While serving an essentialphysiological role, the proteosome is also responsible for theinappropriate or accelerated protein degradation that occurs as a resultor cause of pathological conditions in which normal cellular processesbecome disregulated. One notable example is cancer, in which theunregulated proteosome-mediated degradation of cell cycle regulatoryproteins, including cyclins, cyclin dependent kinase inhibitors, andtumor suppressor genes, results in accelerated and uncontrolled mitosis,thereby promoting cancer growth and spread. (Goldberg et al. 1995 Chem.& Biol. 2:503-508; Coux et al. 1996 Annu. Rev. Biochem. 65:801-847;Deshaies 1995 Trends Cell Biol. 5:428-434). The inhibition of theproteosome enzymatic function holds promise in arresting or blunting thedisease progression in disease states such as cancer or inflammation.

Proteosome inhibitors, e.g., lactacystin and its analogs, have beenshown to block the development of the preerythrocytic and erythrocyticstages of Plasmodium spp, the malaria parasites. During both its hepaticand erythrocytic stages the parasite undergoes radical morphologicalchanges and many rounds of replication, events that likely requireproteosome activity. Lactacystin has been found to covalently modify thecatalytic N-terminal threonines of the active sites of proteosomes,inhibiting the activity of all proteosomes examined, including those inmammalian cells, protozoa, and archeae. (Gantt et al. 1998 AntimicrobAgents Chemother. 42:2731-2738).

The human fibroblast activation protein (FAPα) is a M_(r) 95,000 cellsurface molecule originally identified with monoclonal antibody (mAb)F19 (Rettig et al. 1988 Proc. Natl. Acad. Sci. USA 85:3110-3114; Rettiget al. 1993 Cancer Res. 53:3327-3335). The FAPα cDNA codes for a type IIintegral membrane protein with a large extracellular domain,trans-membrane segment, and short cytoplasmic tail (Scanlan et al. 1994Proc. Natl. Acad. Sci. USA 91:5657-5661; WO 97/34927). FAPα shows 48%amino acid sequence identity to the T-cell activation antigen CD26, alsoknown as dipeptidyl peptidase IV (DPP IV), a membrane-bound protein withdipeptidyl peptidase activity (Scanlan et al.). FAPα has enzymaticactivity and is a member of the serine protease family, with serine 624being critical for enzymatic function (WO 97/34927). Work using amembrane overlay assay revealed that FAPα dimers are able to cleaveAla-Pro-7-amino-4-trifluoromethyl coumarin,Gly-Pro-7-amino-4-trifluoromethyl coumarin, andLys-Pro-7-amino-4-trifluoromethyl coumarin dipeptides (WO 97/34927).

FAPα is selectively expressed in reactive stromal fibroblasts of manyhistological types of human epithelial cancers, granulation tissue ofhealing wounds, and malignant cells of certain bone and soft tissuesarcomas. Normal adult tissues are generally devoid of detectable FAPα,but some foetal mesenchymal tissues transiently express the molecule. Incontrast, most of the common types of epithelial cancers, including >90%of breast, non-small-cell lung, and colorectal carcinomas, containFAPα-reactive stromal fibroblasts (Scanlan et al.). These FAPα⁺fibroblasts accompany newly formed tumor blood vessels, forming adistinct cellular compartment interposed between the tumor capillaryendothelium and the basal aspect of malignant epithelial cell clusters(Welt et al. 1994 J. Clin. Oncol. 12(6):1193-1203). While FAPα⁺ stromalfibroblasts are found in both primary and metastatic carcinomas, thebenign and premalignant epithelial lesions tested (Welt et al.), such asfibroadenomas of the breast and colorectal adenomas, only rarely containFAPα⁺ stromal cells. Based on the restricted distribution pattern ofFAPα in normal tissues and its uniform expression in the supportingstroma of many malignant tumors, clinical trials with ¹³¹I-labeled mAbF19 have been initiated in patients with metastatic colon carcinomas(Welt et al.)

SUMMARY OF THE INVENTION

The present invention relates to prodrugs of protease inhibitors, suchas inhibitors of proteosome, DPP IV, FAPα and the like. These“pro-inhibitors” are activated, i.e., cleaved by an “activatingprotease” to release an active inhibitor moiety in proximity to a“target protease”. The identity of activating protease and targetprotease can be the same (such pro-inhibitors being referred to as“Target-Activated Smart Protease Inhibitors” or “TASPI”) or different(e.g., “Target-Directed Smart Protease Inhibitors” or “TDSPI”). Afteractivation of the pro-inhibitor, the active inhibitor moiety canself-inactivate by, e.g., intramolecular-cyclization or cis-transisomerization.

These pro-inhibitors of the present invention exhibit surprisingcharacteristics including improved potency, extended duration of action,improved stability, and/or a decrease in toxicity.

In certain preferred embodiments, the present invention providespro-inhibitors which inhibit post-proline cleaving enzymes, such asinhibitors of dipeptidyl peptidase IV (DPP IV), as well aspharmaceutical compositions thereof, and methods for using suchinhibitors. Such pro-inhibitors of the present invention can be used aspart of treatments for a variety of disorders/conditions, such as thosewhich are mediated by DPP IV. For instance, the subject inhibitors canbe used to up-regulate GIP and GLP-1 activities, e.g., by increasing thehalf-life of those hormones, as part of a treatment for regulatingglucose levels and/or metabolism, e.g., to reduce insulin resistance,treat hyperglycemia, hyperinsulinemia, obesity, hyperlipidemia,hyperlipoprotein-emia (such as chylomicrons, VLDL and LDL), and toregulate body fat and more generally lipid stores, and, more generally,for the improvement of metabolism disorders, especially those associatedwith diabetes, obesity and/or atherosclerosis.

While not wishing to bound by any particular theory, it is observed thatcompounds which inhibit DPP IV are, correlatively, able to improveglucose tolerance, though not necessarily through mechanisms involvingDPP IV inhibition per se. Indeed, the applicant has previouslydemonstrated an effect in mice lacking a GLP-1 receptor suggesting thatthe subject method may not include a mechanism of action directlyimplicating GLP-1 itself, though it has not been ruled out that GLP-1may have other receptors. However, in light of the correlation with DPPIV inhibition, in preferred embodiments, the subject method utilizes anagent with a Ki for DPP IV inhibition of 50.0 nm or less, morepreferably of 10.0 nm or less, and even more preferably of 1.0, 0.1 oreven 0.01 nM or less. Indeed, inhibitors with Ki values in the picomolarand even femtomolar range are contemplated. Thus, while certain of thepro-inhibitors described herein, for convenience, as “DPP IVinhibitors”, it will be understood that such nomenclature is notintending to limit the subject invention to a particular mechanism ofaction.

Certain of the subject compounds have extended duration. Accordingly, incertain preferred embodiments, the inhibitor(s) is selected, and theamount of inhibitor formulated, to provide a dosage which inhibits serumDPP IV levels by at least 50 percent for at least 4 hours after a singledose, and even more preferably for at least 8 hours or even 12 or 16hours after a single dose.

For instance, in certain embodiments the method involves administrationof a DPP IV pro-inhibitor, preferably at a predetermined time(s) duringa 24-hour period, in an amount effective to improve one or more aberrantindices associated with glucose metabolism disorders (e.g., glucoseintolerance, insulin resistance, hyperglycemia, hyperinsulinemia andType I and II diabetes).

In other embodiments, the method involves administration of a DPP IVpro-inhibitor in an amount effective to improve aberrant indicesassociated with obesity. Fat cells release the hormone leptin, whichtravels in the bloodstream to the brain and, through leptin receptorsthere, stimulates production of GLP-1. GLP-1, in turn, produces thesensation of being full. The leading theory is that the fat cells ofmost obese people probably produce enough leptin, but leptin may not beable to properly engage the leptin receptors in the brain, and so doesnot stimulate production of GLP-1. There is accordingly a great deal ofresearch towards utilizing preparations of GLP-1 as an appetitesuppressant. The subject method provides a means for increasing thehalf-life of both endogenous and ectopically added GLP-1 in thetreatment of disorders associated with obesity.

In a more general sense, the present invention provides methods andpro-inhibitor compositions for altering the pharmokinetics of a varietyof different polypeptide hormones by inhibiting the proteolysis of oneor more peptide hormones by DPP IV or some other proteolytic activity.Post-secretory metabolism is an important element in the overallhomeostasis of regulatory peptides, and the other enzymes involved inthese processes may be suitable targets for pharmacological interventionby the subject method.

For example, the subject method can be used to increase the half-life ofother proglucagon-derived peptides, such as glicentin (corresponding toPG 1-69), oxyntomodulin (PG 33-69), glicentin-related pancreaticpolypeptide (GRPP, PG 1-30), intervening peptide-2 (IP-2, PG111-122amide), and glucagon-like peptide-2 (GLP-2, PG 126-158).

GLP-2, for example, has been identified as a factor responsible forinducing proliferation of intestinal epithelium. See, for example,Drucker et al. (1996) PNAS 93:7911. The subject DPP IV pro-inhibitorscan be used as part of a regimen for treating injury, inflammation orresection of intestinal tissue, e.g., where enhanced growth and repairof the intestinal mucosal epithelial is desired, such as in thetreatment of Chron's disease or Inflammatory Bowel Disease (IBD).

DPP IV has also been implicated in the metabolism and inactivation ofgrowth hormone-releasing factor (GHRF). GHRF is a member of the familyof homologous peptides that includes glucagon, secretin, vasoactiveintestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitaryadenylate cyclase activating peptide (PACAP), gastric inhibitory peptide(GIP) and helodermin. Kubiak et al. (1994) Peptide Res 7:153. GHRF issecreted by the hypothalamus, and stimulates the release of growthhormone (GH) from the anterior pituitary. Thus, the subject method canbe used to improve clinical therapy for certain growth hormone deficientchildren, and in clinical therapy of adults to improve nutrition and toalter body composition (muscle vs. fat). The subject method can also beused in veterinary practice, for example, to develop higher yield milkproduction and higher yield, leaner livestock.

Likewise, the DPP IV pro-inhibitors of the subject invention can be usedto alter the plasma half-life of secretin, VIP, PHI, PACAP, GIP and/orhelodermin. Additionally, the subject method can be used to alter thepharmacokinetics of Peptide YY and neuropeptide Y, both members of thepancreatic polypeptide family, as DPP IV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity.

In other embodiments, the subject DPP IV pro-inhibitors can be used tostimulate hematopoiesis.

In still other embodiments, the subject DPP IV pro-inhibitors can beused to inhibit growth or vascularization of transformed cells/tissues,e.g., to inhibit cell proliferation such as that associated with tumorgrowth and metastasis, and for inhibiting angiogenesis in an abnormalproliferative cell mass.

In yet other embodiments, the subject DPP IV pro-inhibitors can be usedto reduce immunological responses, e.g., as an immunosuppressant.

In yet other examples, the DPP IV pro-inhibitors according to thepresent invention can be used to treat CNS maladies such as strokes,tumors, ischemia, Parkinson's disease, memory loss, hearing loss, visionloss, migraines, brain injury, spinal cord injury, Alzheimer's diseaseand amyotrophic lateral sclerosis (which has a CNS component).Additionally, the DPP IV pro-inhibitors can be used to treat disordershaving a more peripheral nature, including multiplesclerosis anddiabetic neuropathy.

Another aspect of the present invention relates to pharmaceuticalcompositions of the subject post-proline cleaving enzyme inhibitors,particularly DPP IV pro-inhibitors, and their uses in treating and/orpreventing disorders which can be improved by altering the homeostasisof peptide hormones. In a preferred embodiment, such DPP IVpro-inhibitors have hypoglycemic and antidiabetic activities, and can beused in the treatment of disorders marked by aberrant glucose metabolism(including storage). In particular embodiments, the compositions of thesubject methods are useful as insulinotropic agents, or to potentiatethe insulinotropic effects of such molecules as GLP-1. In this regard,certain embodiments of the present compositions can be useful for thetreatment and/or prophylaxis of a variety of disorders, including one ormore of: hyperlipidemia, hyperglycemia, obesity, glucose toleranceinsufficiency, insulin resistance and diabetic complications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Structures of AlaboroPro and ProboroPro in alignment withCD26/DPP IV cleavable peptide substrate.

FIG. 2 pH dependent conformational equilibrium of Xaa-boroPro's. Openchain form is favored at low pH values, cyclic structure is favored athigh pH values.

FIGS. 3 a-d pH dependence of DPP IV inhibition by Val-boroPro (a) andAla-boroPro (c) compared to the pH independence of the correspondingsmart drugs (b and d) Inhibitors were incubated overnight in pH 2 or pH8 solutions and then added directly to an enzyme assay solution at pH8.0.

FIG. 4 Examples of pH-dependent conformational equilibrium for differentelectron deficient functional groups.

FIG. 5 Cyclohexylglycine-Proline (or Proline analog)-Valine-boroProline.

FIG. 6 Serum DPP IV activity in rats as a function of time followingthree different doses of CHG-Pro-Val-boroPro given orally. Resultsdemonstrate that CHG-Pro-Val-boro-Pro is orally active.

FIG. 7 Serum DPP IV activity in rats as a function of time followingthree different doses of CHG-Pro-Val-boroPro given subcutaneously.

FIG. 8 Serum DPP IV activity in rats as a function of time following asingle oral dose of 0.025 mg/kg. Each bar represents the average of fourrats. Results demonstrate that CHG-Pro-Val-boroPro outperformsVal-boroPro in inhibition serum DPP IV enzyme activity. Note that withCHG-Pro-Val-boroPro inhibition, although a little slower to develop, isgreater and longer lasting than with Val-boroPro. Results also show thatthe prodrug form of Ala-boroPro, CHG-Pro-Ala-boroPro, chemically verysimilar to CHG-Pro-Val-boroPro, is much less orally active.

FIG. 9 Oral glucose tolerance test (OGTT) in DB/DB mice given four hourspost oral dose of test agents at 0.5 mg/kg Val-boroPro. Dose ofCHG-Pro-Val-boroPro and CHG-Pro-Ala-boroPro calculated to be equivalentto 0.05 mg/kg of Val-boroPro.

FIG. 10 Serum DPP IV activity in rats as a function of time at 1 hr and4 hr intervals following a single oral dose of 0.05 mg/kg. Each barrepresents the average of four rats.

FIG. 11 Structure of Millenium's LDP 341 Proteosome Protease Inhibitor.

FIGS. 12 a,b IC₅₀ concentration graphs of Pro-boro-Pro (a) andCBZ-Ala-boro-Pro (b).

FIGS. 13 a,b IC₅₀ pH dependance of 4-aminomethylbenzylboronic acid (a)and ethyl Gly-boro-Pro(b).

FIGS. 14 a,b IC₅₀ pH dependance of propyl Gly-boroPro (a) and t-butylGly-boroPro (b).

FIG. 15 IC₅₀ concentration graph of Pro-boro-Pro.

FIG. 16 Inhibition profile of AspProGlyboroPro against DPP IV atselected pHs

FIG. 17 Inhibition profile of Suc-AlaAlaProPheAlaboroPro againstchymotrpsin at selected pHs

FIGS. 18 a-c Inhibition profile of HisAlaAspboroPro against DPP IV atselected pHs at different time intervals.

FIGS. 19 a-g Time resolved inhibitory potency of PheProAlaboroProagainst DPP IV at selected pHs at different time intervals.

FIG. 20 Inhibition profile of LysProPheboroLeu against DPP IV atselected pHs.

FIG. 21 Inhibition profile of TyrProSerboroPro against DPP IV atselected pHs.

FIGS. 22 a-g Inhibition profile of ChgAla-tBugboroPro against DPP IV atselected pHs at different time intervals.

FIG. 23 Inhibition profile of TyrProTyrProPheboroLeu against DPP IV.

FIGS. 24 a-f Inhibition profile of Gly[[p]]Pro-N-Me-Gly-BoroPro againstDPP IV at selected pHs at different time intervals.

FIG. 25 Inhibition profile of TyrProPheAlaboroPro against DP IV atselected pHs.

FIG. 26 Inhibition profile of tBugProAlaboroPro against DP IV atselected pHs.

FIG. 27 Inhibition profile of ChgProChgboroPro against DP IV at selectedpHs.

FIG. 28 Inhibition profile of Beta EBP against DP IV at selected pHs.

FIG. 29 Inhibition profile of ChgHypEtgboroPro against DP IV at selectedpHs.

FIG. 30 Inhibition profile of ChgPipEtgboroPro against DP IV at selectedpHs.

FIG. 31 Inhibition profile of ChgAzeEtgboroPro against DP IV at selectedpHs.

FIGS. 32 a-g Inhibition profile of ChgThz2EtgboroPro against DP IV atselected pHs at different time intervals.

FIGS. 33 a-f Inhibition profile of ChgThz4EtgboroPro against DP IV atselected pHs at different time intervals.

FIG. 34 Inhibition profile of NH3-ChgProAbuN(me)boroGly against DP IV atselected pHs.

FIGS. 35 a-f Inhibition profile of HisAlaEtgboroPro against DP IV atselected pHs at different time intervals.

FIGS. 36 a-g Inhibition profile of ChgAib-EthylGly-boroPro against DP IVat selected pHs at different time intervals.

FIGS. 37 a-g Inhibition profile of ChgPro-tBug-boroAla against DP IV atselected pHs at different time intervals.

FIGS. 38 a-g Inhibition profile of ChgCpg-EthylGly-boroPro against DP IVat selected pHs at different time intervals.

FIG. 39 Inhibition profile of ChgAla-Etg-boroPro against DP IV atselected pHs.

FIGS. 40 a-g Inhibition profile of Tyr-(D)-ProPheboroPro against DP IVat selected pHs at different time intervals.

FIGS. 41 a-g Inhibition profile of ChgPro-tBug-boroPro against DP IV atselected pHs at different time intervals.

FIG. 42 Inhibition profile of PhePro-Ala-boroPro against DP IV atselected pHs.

FIG. 43 Inhibition profile of TyrProAlaboroPro against DP IV at selectedpHs.

FIG. 44 Inhibition profile of ProProProboroPro against DP IV at selectedpHs.

FIG. 45 Inhibition profile of AlaProProboroPro against DP IV at selectedpHs.

FIG. 46 Inhibition profile of GlyProProboroPro against DP IV at selectedpHs.

FIG. 47 Smart drug compounds.

FIG. 48 Smart drug compounds.

FIG. 49 Smart drug compounds.

FIG. 50 Smart drug compounds.

FIGS. 51 a-c In vivo time resolved inhibition of DPP IV byChgProAlaboroPro, N-Me-PheProAlaboroPro and SarDhpAlaboroPro atdifferent dosage levels.

FIGS. 52 a,b In vivo time resolved inhibition of DPP IV byPheProAlaboroPro at different dosage levels.

FIG. 53 Blood glucose levels in diabetic mice following treatment withPheProAlaboroPro.

FIG. 54 Inhibition profile of N-Me-PheProAlaboroPro against DPP IV.

FIG. 55 Time resolved inhibition of DPP IV by N-Me-PheProAlaboroPro atselected pHs.

FIGS. 56 a-h. Inhibition profile of SarDhpAlaboroPro against DPP IV atselected pHs at different times.

FIG. 57 In vivo activity of PheProAlaboroPro.

FIG. 58 Increased glucose-stimulated insulin and GLP-1 in DPP IV −/−mice.

FIG. 59 GLP-1 signaling is not required for action of DPP IV inhibitors.

FIG. 60 Comparisons of time resolved potencies of various DPP IVinhibitors in mice.

FIG. 61 Comparison between ChgProValboroPro and ValboroPro in blockingDPP IV catalytic activity in vivo in mice.

FIG. 62 ChgProValboroPro is more potent, longer acting, and safer thanValboroPro in vivo in rats. Each bar represents the average of fouranimals.

FIG. 63 ChgProValboroPro performs better than ValboroPro in oral glucosetolerance tests in db/db mice at a single oral dose of 0.05 mg/kg. Testarticles given three hours before oral glucose challenge at T=0 on abovegraph. Each time point represents the average of five mice.

FIGS. 64 a-d Smart pro-inhibitor molecule versions of potent DPP IVinhibitors exhibit increased potency at high pH and pH independence inin vitro DPP IV inhibition assays.

FIG. 65 ChgProValboroPro outperforms GLP-1 in lowering fasting bloodglucose in db/db mice. A dose of 1.7 mg corresponds to the 0.05 mg/kgdose.

FIG. 66 ChgProValboroPro is as effective as exendin-4 in loweringfasting blood glucose in diabetic mice. Exendin is in phase 3 clinicaltrials for the treatment of type 2 diabetes (Amylin). Exendin-4 iswidely regarded as the most powerful agent currently know for loweringfasting blood glucose levels and for improving glycemic control indiabetics.

FIG. 67 The greater the hyperglycemia the greater is the effectexendin-4 exerts in lowering fasting blood glucose. This shows thatChgProValboroPro does about what the highest dose of exendin-4 could beexpected to do in the db/db mice to which ChgProValboroPro was given.

FIG. 68 Ki values for DPP IV inhibitors. K_(i) values measured in 0.1HEPES pH 8.0, 0.14 M NaCl at 23 C with 200 mM AlaPro-P-nitroanalide asthe substrate. The numbers in the parentheses are the standard errors.

FIG. 69 Activity profiles for selected DPP IV inhibitors. K_(i) valuesmeasured in 0.1 HEPES pH 8.0, 0.14 M NaCl at 23 C with 200 mMAlaPro-P-nitroanalide as the substrate. The numbers in the parenthesesare the standard errors.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

Dipeptide boronic acid inhibitors of the type Xaa-boroPro, where Xaarefers to any natural or non-naturally occurring amino acid, and boroProrefers to the analog of proline in which the C-terminal carboxylate hasbeen replaced by a boronyl group (FIG. 1), are very potent inhibitors ofdipeptidyl amino peptidase type IV (DPP IV). However, these inhibitorsundergo a pH dependent conformational equilibration between an openchain conformer and cyclic conformer (FIG. 2). The open chain formpredominates under acidic condition while the cyclic form predominatesat neutral and basic conditions. The open chain form is the one activeas an enzyme inhibitor; the cyclic form is substantially inactive as anenzyme inhibitor. Similar reversible intraconversions occur with otherdipeptide inhibitors, such as nitriles, aldehydes, etc, when thedipeptide includes a free amino terminus. The net result of thecyclization reaction, which can occur at physiological pH's, is toreduce the effectiveness, potency and duration of action of thesemolecules as enzyme inhibitors and also therefore decrease theirattractiveness as potential drugs.

The rates of inter-conversion between open and cyclic forms, and theequilibrium constant (K_(eg)=[cyclic]/[open]) depend in part on pH. Forexample a boroAlanine, boroLeucine, boroArginine, or any natural ornon-naturally occurring amino acid having a boronyl group in place ofits C-terminal carboxylate will also undergo cyclization whenincorporated into dipeptide or dipeptide-like structures (such asNH₂-Xaa-boroAla, NH₂-Xaa-boroLeu, NH₂-Xaa-boroArg), although theintrinsic rates and equilibrium position will differ. The T_(1/2) forthe inter-conversion reaction may vary from a few seconds to minuteswhen boroXaa is boroGly or boroAla, to hours when it is boroPro.(Pro-boroPro has T_(1/2) of 2 & 8 hours respectively for the cyclizationand uncyclization reactions). The equilibrium constant K_(eq) can varyfrom 0.01 to 0.001 (in favor of the open conformer) at pH 2.0 to 100 to1000 (in favor of the cyclic conformer) at pH 7.2 (i.e., physiologicalpH). FIG. 3 illustrates this phenomenon for Ala-boroPro, Pro-boroPro andVal-boroPro as it demonstrates that all three molecules are moreeffective as inhibitors of DPP IV when pre-incubated at low pH than athigh pH. Importantly, the molecules have a relatively low rates ofdecomposition, even under conditions in which the inactive conformer ispresent as the predominates. The inhibitory activity of the open chainconformer can be restored if the molecules incubated at high pH arere-equilibrated in low pH buffer for a time sufficient to re-establishthe low pH equilibrium. The time required to restore full activityvaries from several hours to several days.

In certain embodiments of the present invention, dipeptide anddipeptide-like transition state analogue inhibitors with other electrondeficient functional groups in place of the boronyl group will alsoexhibit similar reversible cyclization reactions with consequentattenuation of their potency. These include, for example nitriles (CN),aldehydes (CHO), trifluoromethylketones (COCF₃), and alpha keto amides(COCONH₂) (FIG. 4). In addition, the reversible self-inactivationphenomenon may also be attributable, although possibly to a lesserdegree, to cis/trans isomerization, especially for dipeptides anddipeptide-like transition state analogue inhibitors which includeproline at P1 position. Thus, though the details may vary considerablyfrom one electrophilic transition state mimetic to another, thecyclization or cis-trans isomerization reaction can nonetheless beexpected in each case to reduce the effectiveness of the correspondinginhibitors relative to what the effectiveness would be if cyclization orcis-trans isomerization did not occur. The reduction in effectiveness,i.e. the ratio of K_(i)'s of the active inhibitor to theself-inactivated inhibitor, can vary from factors of as little as two orso, to many thousands and even greater.

In certain preferred embodiments of the present invention, thedeficiency, or handicap, of the dipeptide transition state structures asenzyme inhibitors owing to the cyclization reaction is eliminated, whileat the same time making use of the cyclization reaction to furtherimprove the specificity, safety and shell-life of these entities asdrugs. The new molecules of the invention have highly desirableattributes that in combination are unprecedented, and which thereforerepresent a wholly new class of molecules that we term “smart” proteaseinhibitors. These attributes include a non-inhibitory prodrug thatrelease a “hyper”-active inhibitor in the vicinity of the target enzymewhich, when it diffuses away from the target enzyme, will cyclize andtherefore inactivate itself.

II. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

A. Chemical Submoieties

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

‘Acyl’ refers to a group suitable for acylating a nitrogen atom to forman amide or carbamate, a carbon atom to form a ketone, a sulfur atom toform a thioester, or an oxygen atom to form an ester group, e.g., ahydrocarbon attached to a —C(═O)— moiety. Preferred acyl groups includebenzoyl, acetyl, tert-butyl acetyl, pivaloyl, and trifluoroacetyl. Morepreferred acyl groups include acetyl and benzoyl. The most preferredacyl group is acetyl.

The term ‘acylamino’ is art-recognized and preferably refers to a moietythat can be represented by the general formula:

wherein R_(A) and R_(B) each independently represent hydrogen or ahydrocarbon substituent, such as alkyl, heteroalkyl, aryl, heteroaryl,carbocyclic aliphatic, and heterocyclic aliphatic.

The terms ‘amine’ and ‘amino’ are art-recognized and refer to bothunsubstituted and substituted amines as well as quaternary ammoniumsalts, e.g., as can be represented by the general formula:

wherein R_(A), R_(C), and R_(D) each independently represent hydrogen ora hydrocarbon substituent, or R_(A) and R_(C) taken together with the Natom to which they are attached complete a heterocycle having from 4 to8 atoms in the ring structure. In preferred embodiments, none of R_(A),R_(C), and R_(D) is acyl, e.g., R_(A), R_(C), and R_(D) are selectedfrom hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, carbocyclicaliphatic, and heterocyclic aliphatic. The term ‘alkylamine’ as usedherein means an amine group, as defined above, having at least onesubstituted or unsubstituted alkyl attached thereto. Amino groups thatare positively charged (e.g., R_(D) is present) are referred to as‘ammonium’ groups. In amino groups other than ammonium groups, the amineis preferably basic, e.g., its conjugate acid has a pK_(a) above 7.

The terms ‘amido’ and ‘amide’ are art-recognized as an amino-substitutedcarbonyl, such as a moiety that can be represented by the generalformula:

wherein R_(A) and R_(C) are as defined above. In certain embodiments,the amide will include imides.

‘Alkyl’ refers to a saturated or unsaturated hydrocarbon chain having 1to 18 carbon atoms, preferably 1 to 12, more preferably 1 to 6, morepreferably still 1 to 4 carbon atoms. Alkyl chains may be straight(e.g., n-butyl) or branched (e.g., sec-butyl, isobutyl, or t-butyl).Preferred branched alkyls have one or two branches, preferably onebranch. Preferred alkyls are saturated. Unsaturated alkyls have one ormore double bonds and/or one or more triple bonds. Preferred unsaturatedalkyls have one or two double bonds or one triple bond, more preferablyone double bond. Alkyl chains may be unsubstituted or substituted withfrom 1 to 4 substituents. Preferred alkyls are unsubstituted. Preferredsubstituted alkyls are mono-, di-, or trisubstituted. Preferred alkylsubstituents include halo, haloalkyl, hydroxy, aryl (e.g., phenyl,tolyl, alkoxyphenyl, alkyloxycarbonylphenyl, halophenyl), heterocyclyl,and heteroaryl.

The terms ‘alkenyl’ and ‘alkynyl’ refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively.

When not otherwise indicated, the terms alkyl, alkenyl and alkynylpreferably refer to lower alkyl, alkenyl and lower alkynyl groups,respectively, e.g., having from 1-8 carbons.

The terms ‘alkoxyl’ and ‘alkoxy’ as used herein refer to an —O-alkylgroup.

Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy, and the like. An ‘etheR is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of a hydrocarbon thatrenders that hydrocarbon an ether can be an alkoxyl, or another moietysuch as —O-aryl, —O-heteroaryl, —O-heteroalkyl, —O-aralkyl,—O-heteroaralkyl, —O-carbocylic aliphatic, or —O-heterocyclic aliphatic.

An ‘alkylseleno’ or ‘selenoalkyl’ refers to a —Se-alkyl group.‘Selenoethers’ more broadly refers to two hydrocarbon groups linked by aselenium atom. Accordingly, the substituent of a hydrocarbon thatrenders that hydrocarbon a selenoether can be an alkylseleno, or anothermoiety such as —Se-aryl, —Se-heteroaryl, —Se-heteroalkyl, —Se-aralkyl,—Se-heteroaralkyl, —Se-carbocylic aliphatic, or —Se-heterocyclicaliphatic.

The term ‘alkylthio’ refers to an —S-alkyl group. Representativealkylthio groups include methylthio, ethylthio, and the like. ‘ThioetheRrefers to a sulfur atom bound to two hydrocarbon substituents, e.g., anether wherein the oxygen is replaced by sulfur. Thus, a thioethersubstituent on a carbon atom refers to a hydrocarbon-substituted sulfuratom substituent, such as alkylthio or arylthio, etc.

The term ‘aralkyl’, as used herein, refers to an alkyl group substitutedwith an aryl group.

‘Aryl ring’ refers to an aromatic hydrocarbon ring system. Aromaticrings are monocyclic or fused bicyclic ring systems, such as phenyl,naphthyl, etc. Monocyclic aromatic rings contain from about 5 to about10 carbon atoms, preferably from 5 to 7 carbon atoms, and mostpreferably from 5 to 6 carbon atoms in the ring. Bicyclic aromatic ringscontain from 8 to 12 carbon atoms, preferably 9 or 10 carbon atoms inthe ring. The term ‘aryl’ also includes bicyclic ring systems whereinonly one of the rings is aromatic, e.g., the other ring is cycloalkyl,cycloalkenyl, or heterocyclyl. Aromatic rings may be unsubstituted orsubstituted with from 1 to about 5 substituents on the ring. Preferredaromatic ring substituents include: halo, cyano, lower alkyl,heteroalkyl, haloalkyl, phenyl, phenoxy, or any combination thereof.More preferred substituents include lower alkyl, cyano, halo, andhaloalkyl.

‘Biohydrolyzable amide’ refers to an amide moiety that is cleaved (e.g.,to form a hydroxyl and a carboxylic acid) under physiologicalconditions. Physiological conditions include the acidic and basicenvironments of the digestive tract (e.g., stomach, intestines, etc.),enzymatic cleavage, metabolism, and other biological processes, andpreferably refer to physiological conditions in a vertebrate, such as amammal.

‘Biohydrolyzable esteR refers to an ester moiety that is cleaved (e.g.,to form a hydroxyl and a carboxylic acid) under physiologicalconditions. Physiological conditions include the acidic and basicenvironments of the digestive tract (e.g., stomach, intestines, etc.),enzymatic cleavage, metabolism, and other biological processes, andpreferably refer to physiological conditions in a vertebrate, such as amammal.

‘Biohydrolyzable imide’ refers to an imide moiety that is cleaved (e.g.,to form a hydroxyl and a carboxylic acid) under physiologicalconditions. Physiological conditions include the acidic and basicenvironments of the digestive tract (e.g., stomach, intestines, etc.),enzymatic cleavage, metabolism, and other biological processes, andpreferably refer to physiological conditions in a vertebrate, such as amammal.

‘Carbocyclic aliphatic ring’ refers to a saturated or unsaturatedhydrocarbon ring. Carbocyclic aliphatic rings are not aromatic.Carbocyclic aliphatic rings are monocyclic, or are fused, spiro, orbridged bicyclic ring systems. Monocyclic carbocyclic aliphatic ringscontain from about 4 to about 10 carbon atoms, preferably from 4 to 7carbon atoms, and most preferably from 5 to 6 carbon atoms in the ring.Bicyclic carbocyclic aliphatic rings contain from 8 to 12 carbon atoms,preferably from 9 to 10 carbon atoms in the ring. Carbocyclic aliphaticrings may be unsubstituted or substituted with from 1 to 4 substituentson the ring. Preferred carbocyclic aliphatic ring substituents includehalo, cyano, alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or anycombination thereof. More preferred substituents include halo andhaloalkyl. Preferred carbocyclic aliphatic rings include cyclopentyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. More preferredcarbocyclic aliphatic rings include cyclohexyl, cycloheptyl, andcyclooctyl.

The term ‘carbonyl’ is art-recognized and includes such moieties as canbe represented by the general formula:

wherein R_(X) is a bond or represents an oxygen or a sulfur, and R_(E)represents a hydrogen, hydrocarbon substituent, or a pharmaceuticallyacceptable salt, R_(F) represents a hydrogen or hydrocarbon substituent.Where R_(X) is an oxygen and R_(E) or R_(F) is not hydrogen, the formularepresents an ‘esteR. Where R_(X) is an oxygen, and R_(E) is as definedabove, the moiety is referred to herein as a carboxyl group, andparticularly when R_(E) is a hydrogen, the formula represents a‘carboxylic acid’. Where R_(X) is an oxygen, and R_(F) is hydrogen, theformula represents a ‘formate’. In general, where the oxygen atom of theabove formula is replaced by sulfur, the formula represents a‘thiocarbonyl’ group. Where Rx is a sulfur and R_(E) or R_(F) is nothydrogen, the formula represents a ‘thioester.’ Where R_(X) is a sulfurand R_(E) is hydrogen, the formula represents a ‘thiocarboxylic acid.’Where R_(X) is a sulfur and R_(F) is hydrogen, the formula represents a‘thioformate.’ On the other hand, where R_(X) is a bond, R_(E) is nothydrogen, and the carbonyl is bound to a hydrocarbon, the above formularepresents a ‘ketone’ group. Where Rx is a bond, R_(E) is hydrogen, andthe carbonyl is bound to a hydrocarbon, the above formula represents an‘aldehyde’ or ‘formyl’ group.

‘Ci alkyl’ is a heteroalkyl chain having i member atoms. For example, C4alkyls contain four carbon member atoms. C4 alkyls containing may besaturated or unsaturated with one or two double bonds (cis or trans) orone triple bond. Preferred C4 alkyls are saturated. Preferredunsaturated C4 alkyl have one double bond. C4 alkyl may be unsubstitutedor substituted with one or two substituents. Preferred substituentsinclude lower alkyl, lower heteroalkyl, cyano, halo, and haloalkyl.

‘Halogen’ refers to fluoro, chloro, bromo, or iodo substituents.Preferred halo are fluoro, chloro and bromo; more preferred are chloroand fluoro.

‘Haloalkyl’ refers to a straight, branched, or cyclic hydrocarbonsubstituted with one or more halo substituents. Preferred haloalkyl areC1-C12; more preferred are C1-C6; more preferred still are C1-C3.Preferred halo substituents are fluoro and chloro. The most preferredhaloalkyl is trifluoromethyl.

‘Heteroalkyl’ is a saturated or unsaturated chain of carbon atoms and atleast one heteroatom, wherein no two heteroatoms are adjacent.Heteroalkyl chains contain from 1 to 18 member atoms (carbon andheteroatoms) in the chain, preferably 1 to 12, more preferably 1 to 6,more preferably still 1 to 4. Heteroalkyl chains may be straight orbranched. Preferred branched heteroalkyl have one or two branches,preferably one branch. Preferred heteroalkyl are saturated. Unsaturatedheteroalkyl have one or more double bonds and/or one or more triplebonds. Prefer-red unsaturated heteroalkyl have one or two double bondsor one triple bond, more preferably one double bond. Heteroalkyl chainsmay be unsubstituted or substituted with from 1 to about 4 substituentsunless otherwise specified. Preferred heteroalkyl are unsubstituted.Preferred heteroalkyl substituents include halo, aryl (e.g., phenyl,tolyl, alkoxyphenyl, alkoxycarbonylphenyl, halophenyl), heterocyclyl,heteroaryl. For example, alkyl chains substituted with the followingsubstituents are heteroalkyl: alkoxy (e.g., methoxy, ethoxy, propoxy,butoxy, pentoxy), aryloxy (e.g., phenoxy, chlorophenoxy, tolyloxy,methoxyphenoxy, benzyloxy, alkoxycarbonylphenoxy, acyloxyphenoxy),acyloxy (e.g., propionyloxy, benzoyloxy, acetoxy), carbamoyloxy,carboxy, mercapto, alkylthio, acylthio, arylthio (e.g., phenylthio,chlorophenylthio, alkylphenylthio, alkoxyphenylthio, benzylthio,alkoxycarbonylphenylthio), amino (e.g., amino, mono- and di-C1-C3alkylamino, methylphenylamino, methylbenzylamino, C1-C3 alkylamido,carbamamido, ureido, guanidino).

‘Heteroatom’ refers to a multivalent non-carbon atom, such as a boron,phosphorous, silicon, nitrogen, sulfur, or oxygen atom, preferably anitrogen, sulfur, or oxygen atom. Groups containing more than oneheteroatom may contain different heteroatoms.

‘Heteroaryl ring’ refers to an aromatic ring system containing carbonand from 1 to about 4 heteroatoms in the ring. Heteroaromatic rings aremonocyclic or fused bicyclic ring systems. Monocyclic heteroaromaticrings contain from about 5 to about 10 member atoms (carbon andheteroatoms), preferably from 5 to 7, and most preferably from 5 to 6 inthe ring. Bicyclic heteroaromatic rings contain from 8 to 12 memberatoms, preferably 9 or 10 member atoms in the ring. The term‘heteroaryl’ also includes bicyclic ring systems wherein only one of therings is aromatic, e.g., the other ring is cycloalkyl, cycloalkenyl, orheterocyclyl. Heteroaromatic rings may be unsubstituted or substitutedwith from 1 to about 4 substituents on the ring. Preferredheteroaromatic ring substituents include halo, cyano, lower alkyl,heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof.Preferred heteroaromatic rings include thienyl, thiazolyl, oxazolyl,pyrrolyl, purinyl, pyrimidyl, pyridyl, and furanyl. More preferredheteroaromatic rings include thienyl, furanyl, and pyridyl.

‘Heterocyclic aliphatic ring’ is a non-aromatic saturated or unsaturatedring containing carbon and from 1 to about 4 heteroatoms in the ring,wherein no two heteroatoms are adjacent in the ring and preferably nocarbon in the ring attached to a heteroatom also has a hydroxyl, amino,or thiol group attached to it. Heterocyclic aliphatic rings aremonocyclic, or are fused or bridged bicyclic ring systems. Monocyclicheterocyclic aliphatic rings contain from about 4 to about 10 memberatoms (carbon and heteroatoms), preferably from 4 to 7, and mostpreferably from 5 to 6 member atoms in the ring. Bicyclic heterocyclicaliphatic rings contain from 8 to 12 member atoms, preferably 9 or 10member atoms in the ring. Heterocyclic aliphatic rings may beunsubstituted or substituted with from 1 to about 4 substituents on thering. Preferred heterocyclic aliphatic ring substituents include halo,cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or anycombination thereof. More preferred substituents include halo andhaloalkyl. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, hydantoin,oxazoline, imidazolinetrione, triazolinone, quinoline, phthalazine,naphthyridine, quinoxaline, quinazoline, quinoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,phenazine, phenarsazine, phenothiazine, furazan, phenoxazine,pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine,morpholine, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, and the like. Preferred heterocyclic aliphatic ringsinclude piperazyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl andpiperidyl. Heterocycles can also be polycycles.

The term ‘hydroxyl’ means OH.

‘Lower alkyl’ refers to an alkyl chain comprised of 1 to 4, preferably 1to 3 carbon member atoms, more preferably 1 or 2 carbon member atoms.Lower alkyls may be saturated or unsaturated. Preferred lower alkyls aresaturated. Lower alkyls may be unsubstituted or substituted with one orabout two substituents. Preferred substituents on lower alkyl includecyano, halo, trifluoromethyl, amino, and hydroxyl. Throughout theapplication, preferred alkyl groups are lower alkyls. In preferredembodiments, a substituent designated herein as alkyl is a lower alkyl.Likewise, ‘lower alkenyl’ and ‘lower alkynyl’ have similar chainlengths.

‘Lower heteroalkyl’ refers to a heteroalkyl chain comprised of 1 to 4,preferably 1 to 3 member atoms, more preferably 1 to 2 member atoms.Lower heteroalkyl contain one or two non-adjacent heteroatom memberatoms. Preferred lower heteroalkyl contain one heteroatom member atom.Lower heteroalkyl may be saturated or unsaturated. Preferred lowerheteroalkyl are saturated. Lower heteroalkyl may be unsubstituted orsubstituted with one or about two substituents. Preferred substituentson lower heteroalkyl include cyano, halo, trifluoromethyl, and hydroxyl.

‘Mi heteroalkyl’ is a heteroalkyl chain having i member atoms. Forexample, M4 heteroalkyls contain one or two non-adjacent heteroatommember atoms. M4 heteroalkyls containing 1 heteroatom member atom may besaturated or unsaturated with one double bond (cis or trans) or onetriple bond. Preferred M4 heteroalkyl containing 2 heteroatom memberatoms are saturated. Preferred unsaturated M4 heteroalkyl have onedouble bond. M4 heteroalkyl may be unsubstituted or substituted with oneor two substituents. Preferred substituents include lower alkyl, lowerheteroalkyl, cyano, halo, and haloalkyl.

‘Member atom’ refers to a polyvalent atom (e.g., C, O, N, or S atom) ina chain or ring system that constitutes a part of the chain or ring. Forexample, in cresol, six carbon atoms are member atoms of the ring andthe oxygen atom and the carbon atom of the methyl substituent are notmember atoms of the ring.

As used herein, the term ‘nitro’ means NO₂.

‘Phenyl’ is a six-membered monocyclic aromatic ring that may or may notbe substituted with from 1 to 5 substituents. The substituents may belocated at the ortho, meta or para position on the phenyl ring, or anycombination thereof. Preferred phenyl substituents include: halo, cyano,lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combinationthereof. More preferred substituents on the phenyl ring include halo andhaloalkyl. The most preferred substituent is halo.

The terms ‘polycyclyl’ and ‘polycyclic group’ refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, heteroaryls, aryls and/orheterocyclyls) in which two or more member atoms of one ring are memberatoms of a second ring. Rings that are joined through non-adjacent atomsare termed ‘bridged’ rings, and rings that are joined through adjacentatoms are ‘fused rings’.

As used herein, the term “solvate” refers to a complex of variablestoichiometry formed by a solute or a salt or pharmaceuticallyfunctional derivative thereof and a solvent. Such solvents for thepurpose of the invention should not interfere with the biologicalactivity of the solute. Examples of solvents include, but are notlimited to water, methanol, ethanol, and acetic acid. Preferably thesolvent used is a pharmaceutically acceptable solvent. Examples ofpharmaceutically acceptable solvents include water, ethanol, and aceticacid.

The term ‘sulfhydryl’ means SH, and the term ‘sulfonyl’ means —SO₂—.

The term ‘sulfamoyl’ is art-recognized and includes a moiety representedby the general formula:

in which R_(A) and R_(C) are as defined above.

The term ‘sulfate’ is art-recognized and includes a moiety that can berepresented by the general formula:

in which R_(C) is as defined above.

The term ‘sulfonamido’ is art-recognized, and includes a moietyrepresented by the general formula:

in which R_(A) and R_(B) are as defined above.

The terms ‘sulfoxido’ and ‘sulfinyl’, as used herein, are art-recognizedand include a moiety represented by the general formula:

in which R_(A) is as defined above.

A ‘substitution’ or ‘substituent’ on a small organic molecule generallyrefers to a position on a multi-valent atom bound to a moiety other thanhydrogen, e.g., a position on a chain or ring exclusive of the memberatoms of the chain or ring. Such moieties include those defined hereinand others as are known in the art, for example, halogen, alkyl,alkenyl, alkynyl, azide, haloalkyl, hydroxyl, carbonyl (such ascarboxyl, alkoxycarbonyl, formyl, ketone, or acyl), thiocarbonyl (suchas thioester, thioacetate, or thioformate), alkoxyl, phosphoryl,phosphonate, phosphinate, amine, amide, amidine, imine, cyano, nitro,azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,sulfonamido, sulfonyl, silyl, ether, cycloalkyl, heterocyclyl,heteroalkyl, heteroalkenyl, and heteroalkynyl, heteroaralkyl, aralkyl,aryl or heteroaryl. It will be understood by those skilled in the artthat certain substituents, such as aryl, heteroaryl, polycyclyl, alkoxy,alkylamino, alkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, and heteroalkynyl, can themselves besubstituted, if appropriate. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds. It will be understood that ‘substitution’ or ‘substitutedwith’ includes the implicit proviso that such substitution is inaccordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, hydrolysis, etc.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl, and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The terms ortho, mew and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The term “amino-terminal protecting group” as used herein, refers toterminal amino protecting groups that are typically employed in organicsynthesis, especially peptide synthesis. Any of the known categories ofprotecting groups can be employed, including acyl protecting groups,such as acetyl, and benzoyl; aromatic urethane protecting groups, suchas benzyloxycarbonyl; and aliphatic urethane protecting groups, such astert-butoxycarbonyl. See, for example, The Peptides, Gross andMienhoffer, eds., Academic Press, New York (1981), Vol. 3, pp. 3-88; andGreen, T. W. & Wuts, P. G. M., Protective Groups in Organic Synthesis,2nd edition, John Wiley and Sons, Inc., New York (1991). Preferredprotecting groups include aryl-, aralkyl-, heteroaryl- andheteroarylalkyl-carbonyl and sulfonyl moieties.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term ‘hydrocarbon’ is contemplatedto include all permissible compounds or moieties having at least onecarbon-hydrogen bond. In a broad aspect, the permissible hydrocarbonsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivatization with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, or an acidicfunctional group, such as carboxyl, diastereomeric salts can be formedwith an appropriate optically active acid or base, followed byresolution of the diastereomers thus formed by fractionalcrystallization or chromatographic means well known in the art, andsubsequent recovery of the purified enantiomers. Enantiomers may also beseparated using a ‘chiral column’, i.e., by chromatographicallyseparating the enantiomers using chiral molecules bound to a solidsupport.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the sameuseful properties thereof, wherein one or more simple variations ofsubstituents are made which do not adversely affect the efficacy of thecompound. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants that are in themselves known, but are not mentioned here.

B. General Terms

The term “amino acid analog” refers to a compound structurally similarto a naturally occurring amino acid wherein either the C-terminalcarboxy group, the N-terminal amino group or side-chain functional grouphas been chemically modified. For example, aspartic acid-(beta-methylester) is an amino acid analog of aspartic acid; N-ethylglycine is anamino acid analog of glycine; or alanine carboxamide is an amino acidanalog of alanine.

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect.

The terms “gastrointestinal inflammation”, “inflammatory bowel disease”,and “inflammation of the gastrointestinal tract” are usedinterchangeably herein to mean inflammation of any portion of thegastrointestinal tract, from the esophagus to the sigmoid flexure or thetermination of the colon in the rectum. The inflammation can be acute,but, generally, the composition of this invention is used to treatchronic conditions.

The term “healthcare providers” refers to individuals or organizationsthat provide healthcare services to a person, community, etc. Examplesof “healthcare providers” include doctors, hospitals, continuing careretirement communities, skilled nursing facilities, subacute carefacilities, clinics, multispecialty clinics, freestanding ambulatorycenters, home health agencies, and HMO's.

The term “IC₅₀” means the dose of a drug that inhibits a biologicalactivity by 50%.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

A “single oral dosage formulation” is a dosage which provides an amountof drug to produce a serum concentration at least as great as the EC₅₀for that drug, but less than the LD₅₀. Another measure for a single oraldosage formulation is that it provides an amount of drug necessary toproduce a serum concentration at least as great as the IC₅₀ for thatdrug, but less than the LD₅₀. By either measure, a single oral dosageformulation is preferably an amount of drug which produces a serumconcentration at least 10 percent less than the LD₅₀, and even morepreferably at least 50 percent, 75 percent or even 90 percent less thanthe drug's the LD₅₀.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human subject.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the pro-inhibitors of thepresent invention from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) RingeRssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “pharmaceutically acceptable salts” in these instances refersto the relatively non-toxic, inorganic and organic base addition saltsof compounds of the present invention.

The term “pharmaceutically functional derivative” refers to anypharmaceutically acceptable derivative of a pro-inhibitor of the presentinvention, for example, an ester or an amide, which upon administrationto a mammal is capable of providing (directly or indirectly) thepro-inhibitor. Such derivatives are recognizable to those skilled in theart, without undue experimentation. Nevertheless reference is made tothe teaching of Burger's Medicinal Chemistry and Drug Discovery, 5thEdition, Vol 1.

As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable mammalian cell

The term “prodrug” as used herein encompasses compounds that, underphysiological conditions, are converted into therapeutically activeagents. A common method for making a prodrug is to include selectedmoieties that are hydrolyzed under physiological conditions to revealthe desired molecule. In other embodiments, the prodrug is converted byan enzymatic activity of the host animal.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. For instance,the phrase “N-terminal protecting group” or “amino-protecting group” asused herein refers to various amino-protecting groups which can beemployed to protect the N-terminus of an amino acid or peptide againstundesirable reactions. Examples of suitable groups include acylprotecting groups such as, to illustrate, formyl, dansyl, acetyl,benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl; aromaticurethane protecting groups as, for example, benzyloxycarbonyl (Cbz); andaliphatic urethane protecting groups such as t-butoxycarbonyl (Boc) or9-Fluorenylmethoxycarbonyl (FMOC). The field of protecting groupchemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. ProtectiveGroups in Organic Synthesis, 2^(nd) ed.; Wiley: New York, 1991).

The term “reversible conformation-dependent inactivation” refers toconformational changes that occur under physiological conditions, suchas pH-dependent conformation changes, that result in two conformershaving different potency for inhibition of a target protease.Preferably, one of the conformers (the “inactive conformer”) has lessthan 50 percent of the inhibitory activity of active conformers, andeven more preferably less than 25, 10, 5 or even 1 percent. To furtherillustrate, in the case of reversible pH-dependent cyclization, thecyclic conformer preferably has a Ki (inhibitory constant) forinhibiting the target protease at least 5 times greater than the linearconformer, and even more preferably at least 10, 100, 1000 or even10,000 times greater.

The term “shelf-life” typically refers to the time period for which theperformance characteristics of a pro-inhibitor remain at peak. As usedherein, the term “T₉₀” refers to the amount of time it takes for apreparation of the subject pro-inhibitor to degrade to the point that ithas 90 percent of the activity of the starting sample, e.g., adiminishment of 10 percent. Likewise, the term “T₅₀” refers to theamount of time it takes for a preparation of the subject pro-inhibitorto degrade to the point that it has 50 percent of the activity of thestarting sample, e.g., a diminishment of 50 percent. The shelf-life,whether reported as T₉₀ or T₅₀, for a given pharmaceutical preparationof a pro-inhibitor is the measured for the preparation as it is packagedfor use by a healthcare provider or patient.

The term “small” as defined herein refers to a group of 10 atoms orless.

The term “statistically significant” as used herein means that theobtained results are not likely to be due to chance fluctuations at thespecified level of probability. The two most commonly specified levelsof significance are 0.05 (p=0.05) and 0.01 (p=0.01). The level ofsignificance equal to 0.05 and 0.01 means that the probability of erroris 5 out of 100 and 1 out of 100, respectively. With regard to purporteddifferences herein, e.g., improved potency, shelf-life, etc., it will beunderstood that such differences are at least statistically significant.

As used herein the term “substantially soluble” refers to pro-inhibitorswhich can be dissolved in inhalant propeller mixture to form asubstantially clear to hazy solution which will not separate into layersor form a precipitate when left unagitated for a minimum of 24 hours atroom temperature.

By “transdermal patch” is meant a system capable of delivery of a drugto a patient via the skin, or any suitable external surface, includingmucosal membranes, such as those found inside the mouth. Such deliverysystems generally comprise a flexible backing, an adhesive and a drugretaining matrix, the backing protecting the adhesive and matrix and theadhesive holding the whole on the skin of the patient. On contact withthe skin, the drug-retaining matrix delivers pro-inhibitor to the skin,the drug then passing through the skin into the patient's system.

The term “quaternizing agent” refers to a chemical compound whichconverts a nitrogen atom with fewer than four substituents to apositively charged nitrogen atom with four substituents. Examples of“quaternizing agents” include lower alkyl halides, such as methyl,ethyl, propyl, and butyl chloride, bromides, and iodides; dialkylsulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides like benzyl and phenethylbromides, and others.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

A “therapeutically effective amount” of a compound, e.g., such as adipeptidyl peptidase inhibitor of the present invention, with respect tothe subject method of treatment, refers to an amount of the compound(s)in a preparation which, when administered as part of a desired dosageregimen (to a mammal, preferably a human) brings alleviates a symptom,ameliorates a condition, or slows the onset of disease conditionsaccording to clinically acceptable standards for the disorder orcondition to be treated or the cosmetic purpose, e.g., at a reasonablebenefit/risk ratio applicable to any medical treatment.

A “therapeutically effective daily dosage” of a compound, e.g., such asa pro-inhibitor of the present invention, with respect to the subjectmethod of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired daily dosageregimen (to a mammal, preferably a human) brings alleviates a symptom,ameliorates a condition, or slows the onset of disease conditionsaccording to clinically acceptable standards for the disorder orcondition to be treated or the cosmetic purpose, e.g., at a reasonablebenefit/risk ratio applicable to any medical treatment.

III. Exemplary Compounds

In certain embodiments, the subject invention provides “pro-inhibitors”represented by the general formula (I) or a solvate, pharmaceuticallyfunctional derivative or pharmaceutically acceptable salt thereof:

A-G  (I)

wherein

-   -   A represents an “address moiety”, e.g., a peptidyl moiety which        is a substrate for an activating protease;    -   A and G are covalently linked by a bond that is cleaved by the        activating protease; and    -   G represents an “inhibitor moiety”, e.g., which inhibits the        proteolytic activity of a target protease,        wherein, the inhibitor moiety G, when cleaved from A by the        activating serine protease, undergoes reversible        conformation-dependent inactivation (such as intramolecular        cyclization or cis/trans isomerization), and/or inhibits the        target protease with a Ki of 100 nM or less.

In preferred embodiments, the address moiety A represents a C-terminallylinked peptide or peptide analog, e.g., of 2-10 amino acid residues,more preferably 2-4 residues, which is a substrate for the activatingenzyme. In certain preferred embodiments, A is a dipeptidyl ortripepidyl moiety. In certain embodiments, A is derived from naturallyoccurring amino acids or analogs thereof, and in certain preferredembodiments, at least one residue of A is a non-naturally occurringamino acid analog.

In certain preferred embodiments, such as when the address moiety A is asubstrate of DPP IV, the amino terminus of the peptide or peptide analogis blocked with an amino-terminal protecting group, preferably a loweralkyl such as a methyl group.

In preferred embodiments, the inhibitor moiety G is a dipeptidyl moietyand a electrophilic functional group that can form a covalent adductwith a residue in the active site of a protease replacing the carboxylterminus of the dipeptidyl moiety. For instance, the inhibitor moiety Gcan be represented in the general formula (II):

Xaa₁-Xaa₂-W  (II)

wherein

Xaa1 and Xaa2 each independently represent an amino acid residue, e.g.,from naturally occurring amino acids or analogs thereof;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct, as for example, —CN,—CH═NR₅,

-   -   R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,        —(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,        —(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH,        —(CH₂)n-S-alkyl, —(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl,        —(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂, —C(O)C(O)OR₇;    -   R₆ represents, independently for each occurrence, a substituted        or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or        heterocycle;    -   R₇ represents, independently for each occurrence, hydrogen, or a        substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl,        cycloalkyl, cycloalkenyl, or heterocycle; and    -   Y₁ and Y₂ can independently or together be OH, or a group        capable of being hydrolyzed to a hydroxyl group, including        cyclic derivatives where Y₁ and Y₂ are connected via a ring        having from 5 to 8 atoms in the ring structure (such as pinacol        or the like),    -   R₅₀ represents O or S;    -   R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;    -   R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or a        pharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together        with the phosphorous atom to which they are attached complete a        heterocyclic ring having from 5 to 8 atoms in the ring structure    -   X₁ represents a halogen;    -   X₂ and X₃ each represent a hydrogen or a halogen;    -   m is zero or an integer in the range of 1 to 8; and n is an        integer in the range of 1 to 8.

The pro-inhibitors of the present invention do not themselves undergocyclization or other forms of reversible conformation-dependentinactivation, and can be constructed such that they do not inhibit theselected target enzyme, or other enzymes to any significant extent,before being cleaved by the activating protease. That is, thepro-inhibitors are themselves inactive, but produce an active conformerof the inhibitor moiety G in the body when the address moiety A isremoved pro-inhibitor.

However, the molecules of the invention are more than just prodrugs asconventionally understood and defined. The “active” component of aconventional prodrug, when released from the precursor prodrug, does notdiffer chemically or functionally from the “active” component as itwould be prepared in a non-prodrug embodiment. In contrast, theinhibitor moieties G, when released from the pro-inhibitor A-G, are allpredominantly found as the active conformer.

For example, an inhibitory moiety that is subject to reversiblepH-dependent inactivation is produced as the open chain conformer, eventhough it is released within the body at physiological pH (i.e., ˜7.2),a pH that would normally strongly favor the cyclic inactive conformer.If released in the vicinity of the target enzyme, as is intended by thepro-inhibitor design, the inhibitor moieties G will have a higherapparent potency relative to the inhibitor moiety administered alone, asthe latter route of administration of the inhibitor will result insubstantially greater amounts of the inactive cyclic conformer as aconsequence to the equilibration between the active and inactiveconformers at physiological pH. This is especially true at higher pHvalues, but some amount of cyclic structure is also present even atlower pH values for separately prepared inhibitors G.

A second feature that makes the pro-inhibitor molecules of the currentinvention different from typical prodrugs is that the inhibitor moiety,after being generated in the active conformer near the target, undergoesthe reversible conformation-dependent inactivation over time, e.g., asit diffuses away from the target enzyme, thereby reducing thepossibility of deleterious side effects that may result from inhibitionof enzymes occurring in other parts of the patient. This combination ofbeing released in a “hyper” or “super-active,” open-chain form in thevicinity of the target enzyme together with this “programmed”deactivation mechanism makes the molecules of the invention morespecific, effective, and safer (i.e., having fewer side effects) thanthe inhibitor moiety used on its own.

In certain embodiments, the inhibitor moiety G is a dipeptidyl moiety,e.g., derived from naturally occurring amino acids or amino acidanalogs.

In certain embodiments, the inhibitor moiety G is an inhibitor of atarget protease which, when cleaved from pro-inhibitor by the activatingprotease, inhibits the target protease with a K_(i) of 100 nM (10⁻⁷M) orless, and even more preferably, a Ki less than equal to 25 nM, 10 nM(10⁻⁸M), 1 nM (10⁻⁹M), or 0.1 nM (10⁻¹⁰M). In certain embodiments,K_(i)'s of less than 10⁻¹¹M and even 10⁻¹²M have been measured orestimated for the subject inhibitor moieties.

In certain embodiments, the K_(i) for the inactive conformer is at least5 times greater than the K_(i) for the active conformer of the inhibitormoiety, and even more preferably at least 100, 1000 or even 10,000 timesgreater.

In certain preferred embodiments, the equilibrium constant (K_(eq)) forthe reversible conformation-dependent inactivation, such as acyclization reaction, is at least 5:1 in favor of the inactiveconformer, and even more preferably 10:1, 100:1 or even 1,000:1.

In certain preferred embodiments, the therapeutic index for thepro-inhibitor is at least 2 times greater than the therapeutic index forthe inhibitor moiety alone, and even more preferably 5, 10, 50 or even100 times greater.

For many of the subject pro-inhibitors, another improvement over theinhibitor moiety itself is increased stability in pharmaceuticalpreparations, such as in solution, oils or solid formulations. Suchstability can be expressed in terms of shelf-life. In certain preferredembodiments, the subject pro-inhibitor has a T₉₀ of at least 7 days, andeven more preferably of at least 20, 50, 100 or even 200 days. Incertain preferred embodiments, the subject pro-inhibitor has a T₅₀ of atleast 20 days, and even more preferably of at least 50, 100, 200 or even400 days. In certain preferred embodiments, the subject pro-inhibitorhas a T₉₀ as a solid, single oral dosage formulation of at least 20, 50,100 or even 200 days. In certain preferred embodiments, the subjectpro-inhibitor has a T₉₀ as a liquid, single dosage suspension of atleast 20, 50, 100 or even 200 days.

Preferred pharmaceutical preparations of the subject pro-inhibitors aresubstantially pyrogen-free. For example, in certain preferredembodiments, the endotoxin concentration of the subject preparation, asassayed by the via the gel-clot method (as a limits test with comparisonto the maximum allowed FDA limit, as stated in appendix E of theEndotoxin Guidance), is less than 10 EU/mL or EU/single dosageformulation, and even more preferably less than 5, 1, or even 0.1 EU/mLor EU/single dosage formulation.

In certain embodiments, a single administration of the pro-inhibitor,such as bolus injection, oral dosage or inhaled dosage, can produce asustained in vivo effect, such as to provide a therapeutically effectiveamount (≧ED50 concentration) of the inhibitor moiety G for a period ofat least 4 hours, and even more preferably at least 8, 12 or even 16hours.

In certain preferred embodiments, the released inhibitor moiety G, andparticularly the inactive conformer, has half-life (e.g., relative todecomposition into lower molecular weight fragments and/or irreversibleconformers) in serum or other biologically relevant fluid of greaterthan 10 hours, and even more preferably a half-life greater than 24, 48or 120 hours. Such half-life can be measured by determining, forexample, the amount of active conformer that can be generated when thesample is shifted to conditions that reverse the conformation-dependentinactivation. For instance, a sample incubated at pH 7.2—which favorsthe inactive conformer, can be shifted to low pH to determine therelative levels of inhibitor activity over a period of time.

Formulations of the present invention include those especiallyformulated for oral, buccal, parental, transdermal, inhalation,intranasal, transmucosal, implant, or rectal administration.

In certain preferred embodiments, the subject pro-inhibitors are orallyavailable, and can be provided in the form of solid dosage formulationssuitable for oral administration to a human patient.

In certain preferred embodiments, the subject pro-inhibitors aretransdermally active, and can be provided in the form of topical creamor suspension or a transdermal patch.

Another aspect of the invention provides a pharmaceutical packageincluding one or more of the subject pro-inhibitors, and instructions(written and/or pictorial) describing the administration of theformulation to a patient. Merely to illustrate, exemplary packages areappropriately dosed and include instructions for one or more of:treatment or prophylaxis of metabolic disorders, gastrointestinaldisorders, viral disorders, inflammatory disorders, diabetes, obesity,hyperlipidemia, dermatological or mucous membrane disorders, psoriasis,intestinal distress, constipation, autoimmune disorders,encephalomyelitis, complement mediated disorders, glomerulonepritis,lipodystrophy; tissue damage, psychosomatic, depressive, andneuropsychiatric disorders, HIV infection, allergies, inflammation,arthritis, transplant rejection, high blood pressure, congestive heartfailure, tumors, and stress-induced abortions.

Preferably, the package includes the one or more pro-inhibitors providedas a single oral dosage formulation.

Where the pro-inhibitor includes one more chiral centers, in preferredembodiments, the pro-inhibitor is provided as at least 75 mol percent ofthe eutomer (relative to the distomer) of that pro-inhibitor, and evenmore preferably at least 85, 90, 95 or even 99 mol percent. Generally,the eutomer with the L-enantiomer (with respect to the Cα carbon) of anamino acid or amino acid analog.

In certain embodiments, the pro-inhibitor is a tetrapeptidyl moietyrepresented in the general formula (III):

Xaa′₁-Xaa″₂-Xaa₁-Xaa₂-W  (III)

wherein

Xaa′₁, Xaa″₂, Xaa₁ and Xaa₂ each independently represent an amino acidresidue, e.g., from naturally occurring amino acids or analogs thereof;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct, as for example, —CN,—CH═NR₅,

-   -   R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,        —(CH₂)m-R₆, —(CH₂)n—OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,        —(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH,        —(CH₂)n-S-alkyl, —(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl,        —(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂, —C(O)C(O)OR₇;    -   R₆ represents, independently for each occurrence, a substituted        or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or        heterocycle;    -   R₇ represents, independently for each occurrence, hydrogen, or a        substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl,        cycloalkyl, cycloalkenyl, or heterocycle; and    -   Y₁ and Y₂ can independently or together be OH, or a group        capable of being hydrolyzed to a hydroxyl group, including        cyclic derivatives where Y₁ and Y₂ are connected via a ring        having from 5 to 8 atoms in the ring structure (such as pinacol        or the like),    -   R₅₀ represents O or S;    -   R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;    -   R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or a        pharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together        with the phosphorous atom to which they are attached complete a        heterocyclic ring having from 5 to 8 atoms in the ring structure    -   X₁ represents a halogen;    -   X₂ and X₃ each represent a hydrogen or a halogen;    -   m is zero or an integer in the range of 1 to 8; and n is an        integer in the range of 1 to 8.

In certain preferred embodiments, Xaa′₁ includes an amino-terminalprotecting group.

In certain preferred embodiments, Xaa′₁ is an amino acid analog having atetrasubstituted Cβ carbon, e.g., a carbon having four substituents noneof which is a hydrogen. For instance, Xaa′₁ can be an amino acid analogrepresented in the general formula:

wherein: R₈ and R₉ each independently represent a lower alkyl or ahalogen; R₁₀ represents a lower alkyl, an aryl, a hydroxyl group or—(CH₂)_(m)—COOH; Z represents a hydrogen or an amino terminal protectinggroup; and m=0, 1 or 2. In certain preferred embodiments, R₈ and R₉ eachindependently represents a lower alkyl, more preferably methyl, ethyl orpropyl, and even more preferably a methyl. In certain preferredembodiments, R₁₀ represents a lower alkyl, more preferably methyl, ethylor propyl, and even more preferably a methyl. In other preferredembodiments, R₁₀ represents an aryl, such as phenyl or hydroxyphenyl(preferably para-hydroxy). In yet other preferred embodiments, R₁₀represents a hydroxyl group. In certain preferred embodiments, R₁₀represents —(CH₂)_(m)—COOH, where m=0, 1 or 2, and preferably where m is0 or 1.

In general, the subject pro-inhibitors can be divided into two distincttypes on the basis of whether they are activated by the same, or by adifferent enzyme as the target enzyme of the inhibitor moiety. The firsttype will be referred to as Type 1 or Target-Activated Smart ProteaseInhibitors (TASPI), the second as Type 2 or Target-Directed SmartProtease Inhibitors (TDSPI). Both embodiments of the pro-inhibitorsprovide for the specific delivery of the active component, e.g., in a“hyper-active” form to the targeted enzyme, and for attenuation of theinhibitor activity as the inhibitor moiety diffuses away from the targetenzyme, and therefore for advantages in specificity, potency and safetycompared to the inhibitor moiety itself in pure form.

TDSPIs of the present invention offer the additional prospects fortissue, or cellular specific inhibition of targeted enzymes. In otherwords TDSPIs offer the prospect of inhibiting a given enzyme in onegiven cell or tissue type but not in another. For example, every cell ofthe body contains a proteosome protease complex Inhibition of proteasomefunction has a number of practical therapeutic and prophylacticapplications. However, it is difficult to provide for inhibition ofproteosome activity in a cell- or tissue-type selective manner. Incertain embodiments of the current invention, TDSPIs can be constructedto deliver a proteosome inhibitor moiety in selective manner by using apro-inhibitor having an address moiety for a protease that is expressedin or adjacent to the intended target cells or tissue. To illustrate, itcan be activated by FAP or Prostate Specific Antigen (PSA) and theresulting inhibitor moiety G is an inhibitor of the proteosome.

In preferred embodiments of TDSPIs, the address moiety A is not anefficient substrate for the target protease. For instance, as asubstrate, address moiety A preferably has a turnover number as asubstrate for the target protease of less than 1/second, and even morepreferably less than 0.1/second, 0.001/second or even 0.0001/second.

In certain embodiments of the subject pro-inhibitors, the address moietyis a substrate for an activating protease selected from amongst serineproteases, cysteine proteases and metalloproteases. Likewise, theinhibitor moiety can be an dipeptidyl inhibitor for a target proteaseselected from serine proteases, cysteine proteases and metalloproteases.In certain preferred embodiments, the target protease is a serineproteases.

The pro-inhibitors of the present invention can be designed to work withtarget and activating serine proteases including, but not limited to,dipeptidyl peptidase-11 (DPP-XI), dipeptidyl peptidase IV (DPP IV),dipeptidyl peptidase (DPP VIII), dipeptidyl peptidase 9 (DPP IX),aminopeptidase P, fibroblast activating protein alpha (seprase), prolyltripeptidyl peptidase, prolyl oligopeptidase (endoproteinase Pro-C),attractin (soluble dipeptidyl-aminopeptidase), acylaminoacyl-peptidase(N-acylpeptide hydrolase; fMet aminopeptidase) and lysosomal Pro-Xcarboxypeptidase (angiotensinase C, prolyl carboxypeptidase).

The pro-inhibitors of the present invention can be designed to work withtarget and activating metalloproteases including membrane Pro-Xcarboxypeptidase (carboxypeptidase P), angiotensin-converting enzyme(Peptidyl-dipeptidase A multipeptidase], collagenase I (interstitialcollagenase; matrix metalloproteinase 1; MMP-1; Mcol-A), ADAM 10(alpha-secretase, myelin-associated disintegrin metalloproteinase),neprilysin (atriopeptidase; CALLA; CD10; endopeptidase 24.11;enkephalinase), Macrophage elastase (metalloelastase; matrixmetalloproteinase 12; MMP-12], Matrilysin (matrix metalloproteinase 7;MMP-7), and neurolysin (endopeptidase 24.16; microsomal endopeptidase;mitochondrial oligopeptidase).

In certain preferred embodiments, the activating protease is apost-prolyl cleaving protease, such as selected from the groupconsisting of DPP IV, DPP II, Prolyl oligopeptidase (PO), FibroblastActivating Protein (FAP), and prolyl carboxypeptidase. In certainembodiments where the post-prolyl cleaving protease is an endopeptidase,the amino terminus of A is blocked with an amino-terminal protectinggroup, preferably a lower alkyl such as a methyl group.

In other embodiments, the activating protease is selected from the groupconsisting of thrombin (Factor X), matriptase, falcipain, prostatespecific antigen (PSA), and proteases homologous thereto.

In certain preferred embodiments, the target protease is a post-prolylcleaving protease, such as selected from the group consisting of DPP IV,DPP II, Prolyl oligopeptidase (PO), Fibroblast Activating Protein (FAP),and prolyl carboxypeptidase.

In certain preferred embodiments, the subject pro-inhibitor isrepresented in the general formula (IV):

wherein

-   -   A represents a 4-8 membered heterocycle including the N and the        Cα carbon; W represents a functional group which reacts with an        active site residue of the targeted protease to form a covalent        adduct, as for example, —CN, —CH═NR₅,

-   -   R₁ represents a C-terminally linked peptide or peptide analog        which is a substrate for an activating enzyme;    -   R₂ is absent or represents one or more substitutions to the ring        A, each of which can independently be a halogen, a lower alkyl,        a lower alkenyl, a lower alkynyl, a carbonyl (such as a        carboxyl, an ester, a formate, or a ketone), a thiocarbonyl        (such as a thioester, a thioacetate, or a thioformate), an        amino, an acylamino, an amido, a cyano, a nitro, an azido, a        sulfate, a sulfonate, a sulfonamido, —(CH₂)_(m)—R₆,        —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower        alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₆, —(CH₂)_(m)—SH,        —(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,        —(CH₂)_(n)—S—(CH₂)_(m)—R₆.    -   R₃ represents, independently for each occurrence, a hydrogen or        a substituent which does not conjugate the electron pair of the        nitrogen from which it pends, such as a lower alkyl;    -   R₄ represents hydrogen or a small hydrophobic group such as a        halogen, a lower alkyl, a lower alkenyl, or a lower alkynyl;    -   R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,        —(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,        —(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH,        —(CH₂)n-S-alkyl, —(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl,        —(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂, —C(O)C(O)OR₇;    -   R₆ represents, for each occurrence, a substituted or        unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or        heterocycle;    -   R₇ represents, for each occurrence, hydrogen, or a substituted        or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,        cycloalkenyl, or heterocycle; and    -   Y₁ and Y₂ can independently or together be OH, or a group        capable of being hydrolyzed to a hydroxyl group, including        cyclic derivatives where Y₁ and Y₂ are connected via a ring        having from 5 to 8 atoms in the ring structure (such as pinacol        or the like),    -   R₅₀ represents O or S;    -   R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;    -   R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or a        pharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together        with the phosphorous atom to which they are attached complete a        heterocyclic ring having from 5 to 8 atoms in the ring structure    -   X₁ represents a halogen;    -   X₂ and X₃ each represent a hydrogen or a halogen;    -   m is zero or an integer in the range of 1 to 8; and n is an        integer in the range of 1 to 8.

In certain preferred embodiments, R₂ is absent, or represents a smallhydrophobic group.

In certain embodiments, the protease inhibitor is represented in thegeneral formula (V):

where R₁, R₃, R₄ and W are as defined above, and p is an integer from 1to 3. In certain preferred embodiments, p is 1, and R₃ is a hydrogen ineach occurrence.

In certain preferred embodiments of the subject pro-inhibitor structuresII-V above, W represents:

In certain preferred embodiments, R₅ is a hydrogen or —C(X₁)(X₂)X₃,wherein X_(i) is a fluorine, and X₂ and X₃, if halogens, are alsofluorine.

In certain preferred embodiments of the subject pro-inhibitor structuresIV and V, R₄ is a lower alkyl.

In certain preferred embodiments of the subject pro-inhibitor structuresIV and V, R₄ represents a sidechain of an amino acid residue selectedfrom the group consisting of Gly, Ala, Val, Ser, Thr, Ile and Leu.

In certain preferred embodiments of the subject pro-inhibitor structuresIV and V, R₄ represents a sidechain of an amino acid residue representedin the general formula:

wherein

R_(4a) and R_(4b) each independently represent a hydrogen, lower alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl,carboxyl, carboxamide, carbonyl, or cyano, with the caveat that eitherboth or neither of R_(4a) and R_(4b) are hydrogen;

R_(4c) represents a halogen, an amine, an alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl, carboxyl,carboxamide, carbonyl, or cyano; and

z is zero or an integer in the range of 0 to 3.

In certain preferred embodiments of the subject pro-inhibitor structuresIV and V, R₄ represents a sidechain of an amino acid residue representedin the general formula:

wherein: R₈ and R₉ each independently represent a lower alkyl or ahalogen; R₁₀ represents a lower alkyl, an aryl, a hydroxyl group or—(CH₂)_(m)—COOH. In certain preferred embodiments, R₈ and R₉ eachindependently represents a lower alkyl, more preferably methyl, ethyl orpropyl, and even more preferably a methyl. In certain preferredembodiments, R₁₀ represents a lower alkyl, more preferably methyl, ethylor propyl, and even more preferably a methyl. In other preferredembodiments, R₁₀ represents an aryl, such as phenyl or hydroxyphenyl(preferably para-hydroxy). In yet other preferred embodiments, R₁₀represents a hydroxyl group. In certain preferred embodiments, R₁₀represents —(CH₂)_(m)—COOH, where m=0, 1 or 2, and preferably where m is0 or 1.

In certain preferred embodiments of the subject pro-inhibitor structuresIV and V, R₁ is a peptidyl moiety which is a substrate for apost-proline cleaving enzyme.

IV. Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). Protease inhibitors accordingto the invention may be formulated for administration in any convenientway for use in human or veterinary medicine. In certain embodiments, thecompound included in the pharmaceutical preparation may be activeitself, or may be a prodrug, e.g., capable of being converted to anactive compound in a physiological setting.

Thus, one aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) administration by inhalation, forexample, aerosols, nebulizers, or dry powders; (3) parenteraladministration, for example, by subcutaneous, intramuscular orintravenous injection as, for example, a sterile solution or suspension;(4) topical application, for example, as a cream, ointment or sprayapplied to the skin; (5) ophthalmic administration; or (6) intravaginalor intrarectal administration, for example, as a pessary, cream or foam.However, in certain embodiments the subject compounds may be simplydissolved or suspended in sterile water. In certain embodiments, thepharmaceutical preparation is non-pyrogenic, i.e., does not elevate thebody temperature of a patient.

Another aspect of the invention provides for a method for preparing suchpharmaceutical composition by combining the protease inhibitor prodrugand a pharmaceutically acceptable inert carrier, in a single dosageformulation. Methods of preparing these formulations or compositionsinclude the step of bringing into association a compound of the presentinvention with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” 0 and B books, Corvallis, Ore., U.S.A., 1977).

Medicaments which may be administered in inhalant or aerosolformulations according to the invention include protease inhibitorprodrugs useful in inhalation therapy which may be presented in a formwhich is soluble or substantially soluble in the selected propellantsystem.

The particle size of the particulate medicament should be such as topermit inhalation of substantially all of the medicament into the lungsupon administration of the aerosol formulation and will thus desirablybe less than 20 microns, preferably in the range 1 to 10 microns, e.g. 1to 5 microns. The particle size of the medicament may be reduced byconventional means, for example by milling or micronisation.

The final aerosol formulation desirably contains 0.005-10% w/w,preferably 0.005-5% w/w, especially 0.01-1.0% w/w, of medicamentrelative to the total weight of the formulation.

It is desirable that the formulations of the invention contain nocomponents which may provoke the degradation of stratospheric ozone. Inparticular it is desirable that the formulations are substantially freeof chlorofluorocarbons such as CCl₃F, CCl₂F₂ and CF₃CCl₃. As used herein“substantially free” means less than 1% w/w based upon the propellantsystem, in particular less than 0.5%, for example 0.1% or less.

Administration of medicament may be indicated for the treatment of mild,moderate or severe acute or chronic symptoms or for prophylactictreatment. It will be appreciated that the precise dose administeredwill depend on the age and condition of the patient, the particularparticulate medicament used and the frequency of administration and willultimately be at the discretion of the attendant physician. Whencombinations of medicaments are employed the dose of each component ofthe combination will in general be that employed for each component whenused alone. Typically, administration may be one or more times, forexample from 1 to 8 times per day, giving for example 1, 2, 3 or 4 puffseach time. Preferably, administration may be one time per day.

For administration, the drug is suitably inhaled from a nebulizer, froma pressurized metered dose inhaler or as a dry powder from a dry powderinhaler (e.g. sold as TURBUHALER®) or from a dry powder inhalerutilizing gelatin, plastic or other capsules, cartridges or blisterpacks.

A diluent or carrier, generally non-toxic and chemically inert to themedicament e.g. lactose, dextran, mannitol, glucose or any additivesthat will give the medicament a desired taste, can be added to thepowdered medicament.

The micronized mixture may be suspended or dissolved in a liquidpropellant mixture which is kept in a container that is sealed with ametering valve and fitted into a plastic actuator. The propellants usedmay be halocarbons of different chemical formulae. The most frequentlyused halocarbon propellants are trichlorofluoromethane (propellant 11),dichlorodifluoromethane (propellant 12), dichlorotetrafluoroethane(propellant 114), tetrafluoroethane (propellant 134a) and1,1-difluoroethane (propellant 152a). Low concentrations of a surfactantsuch as sorbitan trioleate, lecithin, disodium dioctylsulphosuccinate oroleic acid may also be used to improve the physical stability.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the protease inhibitorprodrug in the proper medium. Absorption enhancers can also be used toincrease the flux of the protease inhibitor prodrugs across the skin.The rate of such flux can be controlled by either providing a ratecontrolling membrane or dispersing the compound in a polymer matrix orgel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the protease inhibitor prodrug.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated and the particular mode of administration. The amountof active ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

In preferred embodiments, the subject compositions are sterile andpyrogen free.

V. Pharmaceutical Packages and Manufacture

One aspect of the present invention provides a packaged pharmaceuticalcomprising one or more pro-inhibitors of the present inventionformulated in a pharmaceutically acceptable excipient, in associationwith instructions (written and/or pictorial) describing the recommendeddosage and/or administration of the formulation to a patient. Suchinstructions may include details for treating or preventing a diseases,and optionally, warnings of possible side effects and drug-drug ordrug-food interactions.

Another aspect of the invention relates to the use of the subjectpro-inhibitors in the manufacture of a medicament for the treatment of adisorder for which inhibition of the target protease of the inhibitormoiety G provides a therapeutic benefit to a patient. Exemplarydisorders are enumerated below.

Yet another aspect of the invention relates to a method for conducting apharmaceutical business, which includes:

a. manufacturing one or more of the subject pro-inhibitors; and

b. marketing to healthcare providers the benefits of using thepreparation to treat or prevent any of the diseases or indications citedherein.

In certain embodiments, the subject business method can includeproviding a distribution network for selling the preparation. It mayalso include providing instruction material to patients or physiciansfor using the preparation to treat and prevent any of the diseases orindications cited herein.

VI. Methods of Use A. Post-Proline Cleaving Enzymes

Certain embodiments of the subject pro-inhibitors can include inhibitormoieties that provide therapeutic compounds for treatment of disordersin mammals which can be treated (alleviated or reduced) by inhibitionDPP IV, DPP II, Prolyl oligopeptidase (PO), Fibroblast ActivatingProtein (FAP), or prolyl carboxypeptidase activities of the mammal.

In certain embodiments, the subject pro-inhibitors can include ainhibitor moiety for DPP IV. In such embodiments, the subjectpro-inhibitors can be used to regulate the proteolysis of suchpolypeptide factors as GLP-1, GIP, GLP-2, GRP, vasoactive intestinalpeptide, peptide histidine methionine, PYY, substance P, β-casomorphine,NPY, PACAP38, prolactin, chorionic gonadotropin, aprotinin,corticotropin-like intermediate lobe peptide, pituitary adenylylcyclase-activating peptide, (Tyr)melanostatin, LD78β(3-70), RANTES,eotaxin procolipase, enterostatin, vasostatin 1, endomorphin,morphiceptin, stromal cell derived factor, macrophage-derived chemokine,granulocyte chemotactic protein-2, and GHRH/GRF.

Accordingly, DPP IV-directed pro-inhibitors can be used to treat avariety of metabolic, gastrointestinal, viral, and inflammatorydiseases, including, but not limited to, diabetes, obesity,hyperlipidemia, dermatological or mucous membrane disorders, psoriasis,intestinal distress, constipation, autoimmune disorders such asencephalomyelitis, complement mediated disorders such asglomerulonepritis, lipodystrophy, and tissue damage, psychosomatic,depressive, and neuropsychiatric disease such as anxiety, depression,insomnia, schizophrenia, epilepsy, spasm, and chronic pain, HIVinfection, allergies, inflammation, arthritis, transplant rejection,high blood pressure, congestive heart failure, tumors, andstress-induced abortions, for example cytokine-mediated murineabortions.

Certain of the subject DPP IV pro-inhibitors can promote satiety, weightloss, and the antidiabetic effects of GLP-1

Certain of the subject DPP IV pro-inhibitors may be useful for treatingintestinal insufficiencies and mucous membrane disorders.

(i). Regulating Blood Glucose Levels

Certain pro-inhibitors of the present invention have the ability tolower blood glucose levels, to relieve obesity, to alleviate impairedglucose tolerance, to inhibit hepatic glucose neogenesis, and to lowerblood lipid levels and to inhibit aldose reductase. They are thus usefulfor the prevention and/or therapy of hyperglycemia, obesity,hyperlipidemia, diabetic complications (including retinopathy,nephropathy, neuropathy, cataracts, coronary artery disease andarteriosclerosis) and furthermore for obesity-related hypertension andosteoporosis.

Diabetes mellitus is a disease characterized by hyperglycemia occurringfrom a relative or absolute decrease in insulin secretion, decreasedinsulin sensitivity or insulin resistance. The morbidity and mortalityof this disease result from vascular, renal, and neurologicalcomplications. An oral glucose tolerance test is a clinical test used todiagnose diabetes. In an oral glucose tolerance test, a patient'sphysiological response to a glucose load or challenge is evaluated.After ingesting the glucose, the patient's physiological response to theglucose challenge is evaluated. Generally, this is accomplished bydetermining the patient's blood glucose levels (the concentration ofglucose in the patient's plasma, serum or whole blood) for severalpredetermined points in time.

In one embodiment, the present invention provides a method for agonizingthe action of GLP-1. It has been determined that isoforms ofGLP-1(GLP-1(7-37) and GLP-1(7-36)), which are derived frompreproglucagon in the intestine and the hind brain, have insulinotropicactivity, i.e., they modulate glucose metabolism. DPP IV cleaves theisoforms to inactive peptides. Thus, in certain embodiments,inhibitor(s) of the present invention can agonize insulinotropicactivity by interfering with the degradation of bioactive GLP-1peptides.

(ii). Agonism of the Effects of Other Peptide Hormones

In another embodiment, certain of the subject pro-inhibitors can be usedto agonize the activity of peptide hormones, e.g., GLP-2, GIP and NPY.

To illustrate further, the present invention provides a method foragonizing the action of GLP-2. It has been determined that GLP-2 acts asa trophic agent, to promote growth of gastrointestinal tissue. Theeffect of GLP-2 is marked particularly by increased growth of the smallbowel, and is therefore herein referred to as an “intestinotrophic”effect. DPP IV is known to cleave GLP-2 into a biologically inactivepeptide. Thus, in one embodiment, inhibition of DPP IV interferes withthe degradation of GLP-2, and thereby increases the plasma half-life ofthat hormone.

In still other embodiments, the subject method can be used to increasethe half-life of other proglucagon-derived peptides, such as glicentin,oxyntomodulin, glicentin-related pancreatic polypeptide (GRPP), and/orintervening peptide-2 (IP-2). For example, glicentin has beendemonstrated to cause proliferation of intestinal mucosa and alsoinhibits a peristalsis of the stomach, and has thus been elucidated asuseful as a therapeutic agent for digestive tract diseases, thus leadingto the present invention.

Thus, in one aspect, the present invention relates to therapeutic andrelated uses of pro-inhibitors for promoting the growth andproliferation of gastrointestinal tissue, most particularly small boweltissue. For instance, the subject method can be used as part of aregimen for treating injury, inflammation or resection of intestinaltissue, e.g., where enhanced growth and repair of the intestinal mucosalepithelial is desired.

With respect to small bowel tissue, such growth is measured convenientlyas a increase in small bowel mass and length, relative to an untreatedcontrol. The effect of subject inhibitors on small bowel also manifestsas an increase in the height of the crypt plus villus axis. Suchactivity is referred to herein as an “intestinotrophic” activity. Theefficacy of the subject method may also be detectable as an increase incrypt cell proliferation and/or a decrease in small bowel epitheliumapoptosis. These cellular effects may be noted most significantly inrelation to the jejunum, including the distal jejunum and particularlythe proximal jejunum, and also in the distal ileum. A compound isconsidered to have “intestinotrophic effect” if a test animal exhibitssignificantly increased small bowel weight, increased height of thecrypt plus villus axis, or increased crypt cell proliferation ordecreased small bowel epithelium apoptosis when treated with thecompound (or genetically engineered to express it themselves). A modelsuitable for determining such gastrointestinal growth is described byU.S. Pat. No. 5,834,428.

In general, patients who would benefit from either increased smallintestinal mass and consequent increased small bowel mucosal functionare candidates for treatment by the subject method. Particularconditions that may be treated include the various forms of sprueincluding celiac sprue which results from a toxic reaction to -gliadinfrom wheat, and is marked by a tremendous loss of villae of the bowel;tropical sprue which results from infection and is marked by partialflattening of the villae; hypogammaglobulinemic sprue which is observedcommonly in patients with common variable immunodeficiency orhypogammaglobulinemia and is marked by significant decrease in villusheight. The therapeutic efficacy of the treatment may be monitored byenteric biopsy to examine the villus morphology, by biochemicalassessment of nutrient absorption, by patient weight gain, or byamelioration of the symptoms associated with these conditions. Otherconditions that may be treated by the subject method, or for which thesubject method may be useful prophylactically, include radiationenteritis, infectious or post-infectious enteritis, regional enteritis(Crohn's disease), small intestinal damage due to toxic or otherchemotherapeutic agents, and patients with short bowel syndrome.

More generally, the present invention provides a therapeutic method fortreating digestive tract diseases. The term “digestive tract” as usedherein means a tube through which food passes, including stomach andintestine. The term “digestive tract diseases” as used herein meansdiseases accompanied by a qualitative or quantitative abnormality in thedigestive tract mucosa, which include, e.g., ulceric or inflammatorydisease; congenital or acquired digestion and absorption disorderincluding malabsorption syndrome; disease caused by loss of a mucosalbarrier function of the gut; and protein-losing gastroenteropathy. Theulceric disease includes, e.g., gastric ulcer, duodenal ulcer, smallintestinal ulcer, colonic ulcer and rectal ulcer. The inflammatorydisease include, e.g., esophagitis, gastritis, duodenitis, enteritis,colitis, Crohn's disease, proctitis, gastrointestinal Behcet, radiationenteritis, radiation colitis, radiation proctitis, enteritis andmedicamentosa. The malabsorption syndrome includes the essentialmalabsorption syndrome such as disaccharide-decomposing enzymedeficiency, glucose-galactose malabsorption, fractose malabsorption;secondary malabsorption syndrome, e.g., the disorder caused by a mucosalatrophy in the digestive tract through the intravenous or parenteralnutrition or elemental diet, the disease caused by the resection andshunt of the small intestine such as short gut syndrome, cul-de-sacsyndrome; and indigestible malabsorption syndrome such as the diseasecaused by resection of the stomach, e.g., dumping syndrome.

The term “therapeutic agent for digestive tract diseases” as used hereinmeans the agents for the prevention and treatment of the digestive tractdiseases, which include, e.g., the therapeutic agent for digestive tractulcer, the therapeutic agent for inflammatory digestive tract disease,the therapeutic agent for mucosal atrophy in the digestive tract and thetherapeutic agent for digestive tract wound, the amelioration agent forthe function of the digestive tract including the agent for recovery ofthe mucosal barrier function and the amelioration agent for digestiveand absorptive function. The ulcers include digestive ulcers anderosions, acute ulcers, namely, acute mucosal lesions.

The subject method, because of promoting proliferation of intestinalmucosa, can be used in the treatment and prevention of pathologicconditions of insufficiency in digestion and absorption, that is,treatment and prevention of mucosal atrophy, or treatment of hypoplasiaof the digestive tract tissues and decrease in these tissues by surgicalremoval as well as improvement of digestion and absorption. Further, thesubject method can be used in the treatment of pathologic mucosalconditions due to inflammatory diseases such as enteritis, Crohn'sdisease and ulceric colitis and also in the treatment of reduction infunction of the digestive tract after operation, for example, in dampingsyndrome as well as in the treatment of duodenal ulcer in conjunctionwith the inhibition of peristalsis of the stomach and rapid migration offood from the stomach to the jejunum. Furthermore, glicentin caneffectively be used in promoting cure of surgical invasion as well as inimproving functions of the digestive tract. Thus, the present inventionalso provides a therapeutic agent for atrophy of the digestive tractmucosa, a therapeutic agent for wounds in the digestive tract and a drugfor improving functions of the digestive tract which comprise glicentinas active ingredients.

Likewise, certain of the DPP IV pro-inhibitors of the subject inventioncan be used to alter the plasma half-life of secretin, VIP, PHI, PACAP,GIP and/or helodermin. Additionally, the subject method can be used toalter the pharmacokinetics of Peptide YY and neuropeptide Y, bothmembers of the pancreatic polypeptide family, as DPP IV has beenimplicated in the processing of those peptides in a manner which altersreceptor selectivity.

Neuropeptide Y (NPY) is believed to act in the regulation vascularsmooth muscle tone, as well as regulation of blood pressure. NPY alsodecreases cardiac contractility. NPY is also the most powerful appetitestimulant known (Wilding et al., (1992) J Endocrinology 132:299-302).The centrally evoked food intake (appetite stimulation) effect ispredominantly mediated by NPY Y1 receptors and causes increase in bodyfat stores and obesity (Stanley et al., (1989) Physiology and Behavior46:173-177).

According to the present invention, a method for treatment of anorexiacomprises administering to a host subject an effective amount of aninhibitor(s) to stimulate the appetite and increase body fat storeswhich thereby substantially relieves the symptoms of anorexia.

A method for treatment of hypotension comprises administering to a hostsubject an effective amount of an inhibitor(s) of the present inventionto mediate vasoconstriction and increase blood pressure which therebysubstantially relieves the symptoms of hypotension.

DPP IV has also been implicated in the metabolism and inactivation ofgrowth hormone-releasing factor (GHRF). GHRF is a member of the familyof homologous peptides that includes glucagon, secretin, vasoactiveintestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitaryadenylate cyclase activating peptide (PACAP), gastric inhibitory peptide(GIP) and helodermin. Kubiak et al. (1994) Peptide Res 7:153. GHRF issecreted by the hypothalamus, and stimulates the release of growthhormone (GH) from the anterior pituitary. Thus, the subject method canbe used to improve clinical therapy for certain growth hormone deficientchildren, and in clinical therapy of adults to improve nutrition and toalter body composition (muscle vs. fat). The subject method can also beused in veterinary practice, for example, to develop higher yield milkproduction and higher yield, leaner livestock.

B. Proteosome Inhibitors

In other embodiments, the subject pro-inhibitors produce inhibitormoieties that are potent and highly selective proteasome inhibitors andcan be employed to inhibit proteasome function. Inhibition of proteasomefunction has a number of practical therapeutic and prophylacticapplications. However, because the proteosome is ubiquitous to livingcells, there is a desire to provide embodiments of the subjectpro-inhibitor that release a proteasome inhibitor using an addressmoiety that is cleaved at or in proximity to the intended target cells.For instance, the proteosome pro-inhibitors embodiments can includeaddress moieties that are substrates for proteases that are expressed intumors or other cells which are undergoing unwanted proliferation, orexpressed in the tissue surrounding the tumor or other targetproliferating cells. For instance, the address moiety can be a substratefor a protease expressed in the stromal layer adjacent a tumor.

In certain embodiments, the proteosome pro-inhibitors of the presentinvention provide a method of reducing the rate of degradation of p53and other tumor suppressors. Such pro-inhibitors are contemplated aspossessing important practical application in treating cellproliferative diseases, such as cancer, restenosis and psoriasis.

In certain embodiments, proteasome pro-inhibitors can be used to inhibitthe processing of internalized cellular or viral antigens into antigenicpeptides that bind to MHC-I molecules in an animal, and are thereforeuseful for treating autoimmune diseases and preventing rejection offoreign tissues, such as transplanted organs or grafts.

Finally, the present invention relates to the use of proteasomepro-inhibitors for treating specific conditions in animals that aremediated or exacerbated, directly or indirectly, by proteasomefunctions. These conditions include inflammatory conditions, such astissue rejection, organ rejection, arthritis, infection, dermatoses,inflammatory bowel disease, asthma, osteoporosis, osteoarthritis andautoimmune disease such as lupus and multiple sclerosis; cellproliferative diseases, such as cancer, psoriasis and restenosis; andaccelerated muscle protein breakdown that accompanies variousphysiological and pathological states and is responsible to a largeextent for the loss of muscle mass (atrophy) that follows nerve injury,fasting, fever, acidosis, and certain endocrinopathies.

Compounds of the present invention inhibit the growth of cancer cells.Thus, the compounds can be employed to treat cancer, psoriasis,restenosis or other cell proliferative diseases in a patient in needthereof.

By the term “treatment of cancer” or “treating cancer” is intendeddescription of an activity of compounds of the present invention whereinsaid activity prevents or alleviates or ameliorates any of the specificphenomena known in the art to be associated with the pathology commonlyknown as “cancer.” The term “cancer” refers to the spectrum ofpathological symptoms associated with the initiation or progression, aswell as metastasis, of malignant tumors. By the term “tumor” isintended, for the purpose of the present invention, a new growth oftissue in which the multiplication of cells is uncontrolled andprogressive. The tumor that is particularly relevant to the invention isthe malignant tumor, one in which the primary tumor has the propertiesof invasion or metastasis or which shows a greater degree of anaplasiathan do benign tumors.

Thus, “treatment of cancer” or “treating cancer” refers to an activitythat prevents, alleviates or ameliorates any of the primary phenomena(initiation, progression, metastasis) or secondary symptoms associatedwith the disease. Cancers that are treatable are broadly divided intothe categories of carcinoma, lymphoma and sarcoma. Examples ofcarcinomas that can be treated by the composition of the presentinvention include, but are not limited to: adenocarcinoma, acinic celladenocarcinoma, adrenal cortical carcinomas, alveoli cell carcinoma,anaplastic carcinoma, basaloid carcinoma, basal cell carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, renaladinol carcinoma,embryonal carcinoma, anometroid carcinoma, fibrolamolar liver cellcarcinoma, follicular carcinomas, giant cell carcinomas, hepatocellularcarcinoma, intraepidermal carcinoma, intraepithelial carcinoma,leptomanigio carcinoma, medullary carcinoma, melanotic carcinoma,menigual carcinoma, mesometonephric carcinoma, oat cell carcinoma,squamal cell carcinoma, sweat gland carcinoma, transitional cellcarcinoma, and tubular cell carcinoma. Sarcomas that can be treated bythe composition of the present invention include, but are not limitedto: amelioblastic sarcoma, angiolithic sarcoma, botryoid sarcoma,endometrial stroma sarcoma, ewing sarcoma, fascicular sarcoma, giantcell sarcoma, granulositic sarcoma, immunoblastic sarcoma, juxaccordialosteogenic sarcoma, coppices sarcoma, leukocytic sarcoma (leukemia),lymphatic sarcoma (lympho sarcoma), medullary sarcoma, myeloid sarcoma(granulocitic sarcoma), austiogenic sarcoma, periosteal sarcoma,reticulum cell sarcoma (histiocytic lymphoma), round cell sarcoma,spindle cell sarcoma, synovial sarcoma, and telangiectatic audiogenicsarcoma. Lymphomas that can be treated by the composition of the presentinvention include, but are not limited to: Hodgkin's disease andlymphocytic lymphomas, such as Burkitt's lymphoma, NPDL, NML, NH anddiffuse lymphomas.

In other embodiments, certain of the proteosome pro-inhibitors employedin the practice of the present invention are capable of preventing thisactivation of NF-kB. Blocking NF-kB activity is contemplated aspossessing important practical application in various areas of medicine,e.g., inflammation, sepsis, AIDS, and the like.

In certain embodiments, the compounds of the present invention can beformulated in topical form for treatment of skin disorders selected frompsoriasis, dermatitis, Lichen planus, acne, and disorders marked byhyperproliferation of skin cells.

In certain embodiments, the compounds of the present invention can beformulated in topical form for treatment of uncontrolled hair growth.

C. Hematopoietic Agonists

In still another aspect, the present invention provides a method forstimulating hematopoietic cells in culture or in vivo. In certainembodiments, the subject DPP IV pro-inhibitors include an address moietythat is a substrate for a protease that is expressed in bone marrow.

According to one aspect of the invention, a method for stimulatinghematopoietic cells in vitro is provided. The method involves (1)contacting the hematopoietic cells with a sufficient amount of an DPP IVpro-inhibitor to increase the number of hematopoietic cells and/or thedifferentiation of such hematopoietic cells relative to the number anddifferentiation of hematopoietic cells.

One important aspect of the invention involves restoring or preventing adeficiency in hematopoietic cell number in a subject. Such deficienciescan arise, for example, from genetic abnormalities, from disease, fromstress, from chemotherapy (e.g. cytotoxic drug treatment, steroid drugtreatment, immunosuppressive drug treatment, etc.) and from radiationtreatment.

The pro-inhibitors of the invention can be administered alone, or incombination with additional agents for treating the condition, e.g., adifferent agent which stimulates activation or proliferation of saidlymphocytes or hematopoietic cells. For example, the pro-inhibitors canbe administered in conjunction with exogenous growth factors andcytokines which are specifically selected to achieve a particularoutcome. For example, if it is desired to stimulate a particularhematopoietic cell type, then growth factors and cytokines whichstimulate proliferation and differentiation of such cell type are used.Thus, it is known that interleukins-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12,13 and 17 are involved in lymphocyte differentiation. Interleukins 3 and4 are involved in mast cell differentiation. Granulocyte macrophagecolony stimulating factor (GMCSF), interleukin-3 and interleukin-5 areinvolved in the eosinophil differentiation. GMCSF, macrophage colonystimulating factor (MCSF) and IL-3 are involved in macrophagedifferentiation. GMCSF, GCSF and IL-3 are involved in neutrophildifferentiation. GMSCF, IL-3, IL-6, IL-11 and TPO are involved inplatelet differentiation. Flt3 Ligand is involved in dendritic cellgrowth. GMCSF, IL-3, and erythropoietin are involved in erythrocytedifferentiation. Finally, the self-renewal of primitive, pluripotentprogenitor cells capable of sustaining hematopoiesis requires SCF, Flt3Ligand, G-CSF, IL-3, IL-6 and IL-11. Various combinations for achievinga desired result will be apparent to those of ordinary skill in the art.

VII. Exemplary Synthetic Schemes

VIII. Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g., a variegated library of compoundsrepresented above, can be screened rapidly in high throughput assays inorder to identify potential protease inhibitor lead compounds, as wellas to refine the specificity, toxicity, and/or cytotoxic-kinetic profileof a lead compound.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds which maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate physical properties can be done byconventional methods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjectprotease inhibitors. See, for example, Blondelle et al. (1995) TrendsAnal. Chem. 14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899:the Ellman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO94/08051; the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen et al.(1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publicationsWO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCTpublication WO93/20242). Accordingly, a variety of libraries on theorder of about 100 to 1,000,000 or more diversomers of the subjectprotease inhibitors can be synthesized and screened for particularactivity or property.

In an exemplary embodiment, a library of candidate protease inhibitordiversomers can be synthesized utilizing a scheme adapted to thetechniques described in the Still et al. PCT publication WO 94/08051,e.g., being linked to a polymer bead by a hydrolyzable or photolyzablegroup, optionally located at one of the positions of the candidateagonists or a substituent of a synthetic intermediate. According to theStill et al. technique, the library is synthesized on a set of beads,each bead including a set of tags identifying the particular diversomeron that bead. The bead library can then be “plated” with proteases forwhich an inhibitor is sought. The diversomers can be released from thebead, e.g., by hydrolysis.

The structures of the compounds useful in the present invention lendthemselves readily to efficient synthesis. The nature of the structuresof the subject compounds, as generally set forth above, allows the rapidcombinatorial assembly of such compounds. For example, as in the schemeset forth below, an activated aryl group, such as an aryl triflate orbromide, attached to a bead or other solid support can be linked toanother aryl group by performing a Stille or Suzuki coupling with anaryl stannane or an aryl boronic acid. If the second aryl group isfunctionalized with an aldehyde, an amine substituent can be addedthrough a reductive amination. Alternatively, the second aryl groupcould be functionalized with a leaving group, such as a triflate,tosylate, or halide, capable of being displaced by an amine. Or, thesecond aryl group may be functionalized with an amine group capable ofundergoing reductive amination with an amine, e.g., CyKNH₂. Otherpossible coupling techniques include transition metal-mediated aminearylation reactions. The resultant secondary amine can then be furtherfunctionalized by an acylation, alkylation, or arylation to generate atertiary amine or amide which can then be cleaved from the resin orsupport. These reactions generally are quite mild and have beensuccessfully applied in combinatorial solid-phase synthesis schemes.Furthermore, the wide range of substrates and coupling partners suitableand available for these reactions permits the rapid assembly of large,diverse libraries of compounds for testing in assays as set forthherein. For certain schemes, and for certain substitutions on thevarious substituents of the subject compounds, one of skill in the artwill recognize the need for masking certain functional groups with asuitable protecting group. Such techniques are well known in the art andare easily applied to combinatorial synthesis schemes.

Many variations on the above and related pathways permit the synthesisof widely diverse libraries of compounds which may be tested as proteaseinhibitors.

IX. Examples Type 1 or Target-Activated SPIs (TASPIs) Example 1 DPP IVActivated, DPP IV Inhibitor

Dipeptidyl amino peptidase type IV (DPP IV) is a type II membrane boundprotease, distributed widely in the body. It is found, for example, inthe intestine, liver and kidney. It is also found on the surface of CD4+and CD8+ T cells where is it known as CD26. Among other things thisenzyme has been demonstrated to hydrolyze and thereby inactivateglucagon like peptide 1 (GLP-1) and gastric inhibitory polypeptide, orglucose-dependent insulinotropic polypeptide (GIP). The activity indegrading these peptides suggests that DPP IV inhibitors could be usefulfor the treatment of Type 2 diabetes, and DPP IV inhibitors have, infact, been demonstrated to improve glucose tolerance in animalsincluding in animal models of diabetes. DPP IV inhibitors have also beendemonstrated to stimulate the proliferation of hematopoetic stem cells,an activity that also cold prove to have therapeutic application. Themechanism underlying this effect, however is not well understood. In anycase, the Xaa-boroPro's (FIG. 1) are the most potent known inhibitors ofDPP IV, and molecules of this class have been demonstrated to havehematopoetic stem-cell proliferative, and anti-diabetic effects in vivoin animals.

Type 1 SPI's, activated by DPP IV and targeting DPP IV can beconstructed by making the R-A portion of R-A-G a structure recognizedand catalytically acted upon by DPP IV so that it is removed to releasethe DPP IV inhibitory moiety G. A Type 1 SPI (TASPI) for DPP IV wouldhave the general structure shown in 2:

R-A of R-A-G corresponds to NH₂-Xaa₁-Xaa₂ of 2. For DPP IV specificityXaa₂ should preferentially be proline or alanine while Xaa₁ can be anynatural or non naturally occurring amino acid, but most preferentiallywith a free amino group. Xaa₃-Xaa₄-T, for example, can be Xaa-boroPro,Xaa-boroAla, or Xaa-Pro-CN. In general Xaa₄ should preferentially be Proor Ala, Xaa₃ can be any natural or non naturally occurring amino acidwhile T can be boronyl, nitrile, aldehyde, alpha keto amide,trifluoromethy ketone etc.

Working examples of a TASPI's for DPP IV includeCyclohexylglycine-Proline-Valine-boroPro line, (CHG-Pro-Val-boroPro)(5),and Cyclohexylglycine-Proline-Ala-boroProline (CHG-Pro-Ala-boroPro).From our prior work on homo- and hetero-conjugates ( ) we know that theP2 side chain of substrates and inhibitors does not make contact withDPP IV but instead must extend away from the enzyme toward the solvent.Thus, DPP IV will cleave dipeptides off the N-terminal of polypeptidesregardless of what the N-terminal happens to be, even if it is a nonnatural amino acid such as CHG. DPP IV will therefore remove theN-terminal CHG-Pro sequence of 5 to release the potent DPP IV inhibitorVal-boroPro, 7. Importantly, the Val-boroPro released will in the openchain form and therefore more potent per unit mass when released in thevicinity of DPP IV than unmodified Val-boroPro can be when prepared inpure form. Placing a CHG (or other similar non naturally occurring aminoacids) in the P4 position of the SPI confers several advantages. One isresistance to amino peptidases, as they are less likely to recognize andcleave from the N-terminal non natural amino acids. Another isresistance to degradation by other post prolyl peptidases and dipeptidylamino peptidases and therefore improved targeting to DPP IV.

FIG. 3 shows that the SPI versions of Val-boroPro and Ala-boroPro do notexhibit the pH dependence in their inhibition of DPP IV in in vitroenzyme assays characteristic of the corresponding Xaa-boroPro inhibitorsand that their apparent affinities are more similar to that of the lowpH form of the free Xaa-boroPro derivative, confirming that theXaa-boroPro moiety is being released in the open chain and more activeconformation as expected (FIG. 3). It is important to note that thex-axis in FIG. 3 refers to the concentration of the SPI, not theconcentration of the released Xaa-boroPro. It is likely that the amountof the Xaa-boroPro released is substantially lower than the startingconcentration of the SPI, and thus FIG. 3 actually underestimates theactual potency of the released Xaa-boroPro.

Table 1 list a number of SPU forms of Xaa-boroPro's showing thatessentially all exhibit the pH independence except for those which are“poor” DPP IV substrates, eg., those having Ala in the P3 site wherecleavage and activation occur.

FIGS. 6 and 7 shows that CHG-Pro-Val-boroPro is very orally active whileFIG. 8 shows it outperforms Val-boroPro in inhibiting serum DPP IVactivity. Although the inhibition takes a little longer to develop(compare at 0.5 hour timepoint) the total inhibition achieved and thelength of time inhibition is maintained is greater with the SPI form ofVal-boroPro than with Val-boroPro (compare inhibition levels at 2, 4 and8 hours). The SPI form of Val-boroPro also appears to be less toxic thanVal-boroPro. For unknown reason Val-boroPro is quite toxic to rats, butapparently not at all to mice (Table 2). Note that a dose of 0.035 mg/kgkilled four out of four Zucker rats, but that more than 100-fold largerdose (i.e., 3.5 mg/kg) has no noticeable toxic effect in mice. Acorresponding dose of CHG-Pro-Val-boroPro (i.e., equivalent to 0.35mg/kg of Val-boroPro) killed only one of four rats (FIG. 6). Thisindicates that inhibiting DPP IV is itself not the cause of toxicity inrats as greater inhibition of serum DPP IV was achieved withCHG-Pro-Val-boroPro than with Val-boroPro itself, with less toxicity.

Table 3 compares the oral activity in mice of Val-boroPro withCHG-Pro-Val-boroPro and CHG-Pro-Ala-boroPro. The results show that likein rats, CHG-Pro-Val-boroPro also outperforms Val-boroPro in mice.

The db/db mice of Table 3 represent an animal model of Type II diabetes.FIG. 9 shows that CHG-Pro-Val-boroPro markedly outperforms Val-boroProin lowering the glucose excursion following an oral glucose challengeand in aiding the return the excursion to normal.

Thus, the SPI version of Val-boroPro is more effective at inhibiting DPPIV in vivo in rats and in mice, the inhibition is more long lasting, ismarkedly more effective in lowering the area under the curve followingan oral glucose challenge in a diabetic mice model, and appears to beless toxic.

Example 2 FAP Activated, FAP Inhibitor

Fibroblast activating protein is a post proline cleaving serine proteasewith some homology to DPP IV which seems to be found only on cellsimmediately surrounding tumors and in some cases healing wounds. Therehas been speculation that blocking this enzyme's activity could initself be useful in treating some forms of solid cancers. FAP'sspecificity resembles that of DPP IV. FAP will for example cleavevarious dipeptide chromagenic substrates that are also substated for DPPIV such as Xaa-Pro-pNA. However, FAP differs from DPP IV in that it hasendopeptidase activity. It will therefore cleave the above Xaa-Pro-pNasubstrate even when the N-terminus is blocked by a CBZ or acetyl group.DPP IV will not cleave such blocked dipeptides, nor will Xaa-boroProinhibitors work well against DPP IV if the N-terminal is blocked. A FAPactivated, FAP inhibitor can be constructed by making R-A of structureR-A-G of compounds of the present invention specific for FAP, while thesegment G (Xaa-Xaa-T) of R-A-G segment can be most anything that alsoinhibits DPP IV. One such variation that should work is illustrated instructure 8.

CBZ-Val-Pro-Val-boroPro  8

This molecule would not be activated by DPP IV although the Val-boroProreleased by FAP would inhibit DPP IV. However, being liberated in thevicinity of FAP together with the cyclization and inactivation thatwould occur as it diffuses away from the FAP target site would confersubstantion FAP specificity on the released Val-boroPro, or other Gmoiety (Xaa-Xaa-T) used to inhibit FAP.

Example 3 Thrombin Activated, Thrombin Inhibitor (or Factor X)

The mechanism of blood clotting present special difficulties whendesigning anti-coagulates targeting thrombin or Factor 10. Both arenormally present in active form at very low concentrations. However,when needed, the cascade mechanisms of the intrinsic or extrinsicpathways rapidly produce huge concentrations of activated thrombin orFactor Xa. The problem this present for anti coagulates is that toprevent clotting huge excesses of thrombin or Factor Xa inhibitors haveto be given and maintained even when levels of activated thrombin arelow. Not having sufficient thrombin or Factor Xa around to bind, theinhibitors are free to block other trypsin like serine proteases causingunwanted side effects. This problem can be overcome by properly designedSPI molecules of the current invention. In this case, R-A should beconstructed such that thrombin or Factor Xa removes it to release athrombin or factor Xa inhibitor. A specific example of such a moleculeis illustrated in structure 9.

R-Arg-Leu-boroArg  9

Type 2 or Target Directed SPIs

Millennium Pharmaceuticals has developed a proteosome proteaseinhibitor, currently in late stage clinical trials for the treatment ofvarious types of cancer. The drug, previously referred to as PS-341, nowreferred to as LDP-341 is a dipeptide boronic acid inhibitor,R-Phe-boroLeu, as shown in FIG. 11. The R group was added largely toprevent cyclization, thereby increasing potency.

Proteosomes are found in every cell of the body. Their catalyticactivity is essentially for cell viability, inhibition of this enzyme istherefore toxic. The potential of LPD-341 as a cancer therapy depends onrapidly proliferating cancer cells being more susceptible to the toxiceffects of proteosome inhibition than normal cells. The currentinvention includes “smart” versions of proteosome inhibitors whichshould have improved efficacy, reduced side effects, and expand therange of therapeutic uses, to for example infectious diseases. Examples,which belong to the Type 2 or Target Directed class of SPIs are outlinedbelow.

Example 4 FAP Activated, Proteosome Protease Inhibitor

Because FAP is associated only with tumors, an FAP activated proteosomeprotease inhibitor should provide substantial improvements in efficacy,and safety over the currently existing non-prodrug Millennium (andother) proteosome protease inhibitors for the treatment of cancer.Structure 10 gives a example of a chemical structure for a FAPactivated, proteosome protease inhibitor.

CBZ-Ala-Pro-Phe-boroLeu  10

Example 5 DPP IV Activated, Proteosome Protease Inhibitor

Because DPP IV is upregulated on activated T cells, directing aproteosome protease inhibitor to these cells cold be therapeutic usefulin the treatment of various autoimmune and other disorders caused byunwanted but activated population of T cells. One example of a structurefor a DPP IV activated, proteosome inhibitor is given in structure 11.

NH₂-Ala-Pro-Phe-boroLeu  11

Example 6 Prostate Specific Antigen (PSA) Activated, Proteosome ProteaseInhibitor

PSA is a serine protease found in abundance in prostate cells. It has achymotrypsin like specificity and will cleave polypeptides followingPhe, Tyr, Ser, Gln. The most favored cleavage sequence appears toSer-Tyr-Gln↓. One example of an SPI targeting specifically theproteosome protease within prostate cells and useful therefore for thetreatment of prostate cancer is shown is structure 12.

Ac-Ser-Try-Gln-Phe-boroLeu  12

Example 7 Matriptase Activated, Proteosome Protease Inhibitor

Matriptase is a recently discovered serine protease found specificallyon the cell surface of certain types of cancer cells. Its specificityhas not been entirely elucidated but similar to trypsin it cleavesfollowing Arg. One example of a matriptase activated proteosomeinhibitor is illustrated in structure 13.

R-Arg-Phe-boroLeu  13

Example 8 Infected Cell Activated, Proteosome Protease Inhibitor

A number of cellular pathogens encode proteases to perform variousfunctions needed by the pathogen. For example Plasmodium falciparum, themicrobe that causes malaria invades red blood cells and degradeshemoglobin as it primary energy source. It produces a cysteine proteaseswith a cathepsin like specificity called falcipain for the purpose ofdegrading hemoglobin. One example of an falcipain activated proteosomeinhibitor useful for the treatment of malaria is illustrates in 14.

Z-Phe-Arg-Phe-boroLeu  14

A number of pathogens rely on proteases to process their polypeptidegene product into functional proteins. Pathogens producing such‘maturational’ proteases are hepatitis type A, B, and C, and HIV amongothers. SPIs targeting cells infected by these agents can be constructedby adding the correct R-A, recognized and cleaved by the pathogen'sprotease to the dipeptide proteosome inhibitor. The resulting SPI shouldseek out and selectively kill infected cells but not uninfected cells ofthe same type.

Example 9 Evaluation of DPP IV Inhibition Following Single OralAdministration of New Chemical Entities to Male Sprague Dawley Rats

Test Article No. 1: Tyrosine-proline-alanine-proline boronic acid

Appearance: White powder with a molecular weight of 446.31 g/mole

Test Article No. 2: Phenylalanine-proline-alanine-proline boronic acid

Appearance: White powder with a molecular weight of 430.31 g/mole

Test Article No. 3:Alanine-proline boronic acid

Appearance: White powder with a molecular weight of 186.02 g/mole

The test articles were stored at room temperature pending use. The doseformulations were prepared for oral administration in deionized (Type I)water as defined in Study Design (Table 1). Forty-six maleSprague-Dawley rats were received from Charles River Canada(St-Constant, QC, Canada) and acclimated to the animal facilities for atleast 96 hr prior to dose administration. Target room conditions were:temperature: 21±3° C.; humidity: 30 to 70%. There were no deviationsfrom these ranges recorded during study conduct. Photoperiod was 12-hrlight and 12-hr dark with exceptions, as necessary, for dosing andsample collection.

The day prior to dose administration, animal were randomly assigned to 9study groups (4 rats/group) according to Study Design (Table 5). On Day1, and following an overnight fast, animals were weighed (body weightrange: 273-301 g) and were administered their respective formulationorally by gavage at a dose volume of 2 mL/kg. Following doseadministration, blood samples (0.25 to 0.40 mL) were collected from eachanimal by jugular venipuncture under isoflurane anesthesia. Bloodsamples were collected (using lithium heparin as the anticoagulant) atpre-dose and again at, 1, 2, 4, 6, 8 and 24 hr post-dose. Blood sampleswere placed on ice pending centrifugation, (3200 g for 10 min at 4° C.nominal). Following centrifugation, plasma was harvested and stored at−20° C. nominal pending shipment for analysis.

Results

No adverse clinical signs were observed during the conduct of thisstudy, suggesting that single dose of the test articles was essentiallyinnocuous at the dose levels administered.

Example 10 Evaluation of DPP IV Inhibition Following Single OralAdministration of Four Triad Compounds in Male Rats

The objectives of this study were to determine the effects of a singleoral doses of four Triad Pharmaceuticals on the inhibition of the enzymedipeptidyl peptidase IV (DPP IV) in male Sprague-Dawley rats and todetermine its potential toxicity. Three of the test articles werepro-drugs of alanine proline boronic acid. This document constitutes thereport describing the in-life procedures used during the conduct of thisstudy. The assessments of DPP IV activity in plasma samples wereperformed by the Sponsor. Results of these investigations will bereported separately.

Methods Test Articles

Test Article 1: L-2-Chg-PRO-ALA-PRO boronic acid

Amount Received: ca 0.6 mL at 93.75 mg/mL in water

Molecular Weight: 422.33 g/mole

Storage Conditions: −20° C. nominal

Test Article 2: N-Me-Phe-PRO-ALA-PRO boronic acid

Amount Received: ca 0.6 mL at 82.5 mg/mL in water

Molecular Weight: 444.33 g/mole

Storage Conditions: −20° C. nominal

Test Article 3: N-Me-Gly-3,4 dehydroproline-ALA-PRO boronic acid

Amount Received: ca 0.6 mL of 66.67 mg/mL in water

Molecular Weight: 352.19 g/mole

Storage Conditions: −20° C. nominal

A total of 2 vials (each containing ca 0.3 mL) for each of Test Articles1, 2, and 3 were stored at ca −20° C. nominal upon receipt and pendinguse. The dose formulations were prepared by the Department of DMPK ofMDSPS. All formulations were prepared for oral administration indeionized (Type I) water as described in the Study Design of Table 6.Dose formulations for Test Articles 1, 2, and 3, were prepared such toensure administration of specifically targeted dose levels of parentdrug based on a molecular weight for ALA-PRO Boronic acid of 136.02g/mole. Thirty-six male Sprague-Dawley rats were received from CharlesRiver Canada (St-Constant, QC, Canada) and acclimated to the animalfacilities for at least one week prior to dose administration. Targetroom conditions were: temperature: 21±3° C.; relative humidity: 30 to70%. There were no deviations from these ranges during study conduct.Photoperiod was 12-hr light and 12-hr dark with exceptions, asnecessary, for dosing and sample collection.

On the day prior to dose administration, animal were randomly assignedto 12 study groups (3 rats/group) according to the Study Designdescribed in Table 6. On Day 1 (day of dose administration), andfollowing an overnight fast, animals were weighed (body weight range:265-292 g) and were administered their respective formulation orally bygavage at a dose volume of 2 mL/kg. Following dose administration, bloodsamples (0.25 to 0.40 mL) were collected from each animal by jugularvenipuncture under isoflurane anesthesia. Blood samples were collected(using lithium heparin as the anticoagulant) at pre-dose and again at 1,2, 4, 6, 8 and 24 hr post-dose. Blood samples were placed on wet icepending centrifugation at 3200 g for 10 min at 4° C. nominal Followingcentrifugation, plasma was harvested and stored at −20° C. nominalpending shipment, on dry ice, for analysis.

Results

Adverse clinical signs and/or mortality were occasionally observed insome animals of Group Nos.: 3, 5, 6, 8, and 9 Animal Nos.: 3001 (Group3) and 8001 (Group 8) were humanely sacrificed because of deterioratingconditions at ca 5 and 4 hr post-dose, respectively. Prior to sacrifice,these animals exhibited, in order, redness of the ears and paws,decreased activity and labored respiration. These signs were observedfor the first time at ca 2 hr post-dose. Moreover, animal No. 9001(Group 9) was found dead at ca 7 hr post-dose. No clinical signs wereobserved on this animal prior to death. Other major clinical signs aresummarized in Table 7.

All patents, applications, and published references cited above arehereby incorporated by reference in their entirety.

TABLE 1 Tetrapeptide SPI forms of several Xaa-boroPro inhibitors of DPPIV and their High and low pHKi values, showing all are essentially pHindependend except for those Ala in P3 (corresponding to the DPP IVcleavage and activation site). This is because these Are not such goodDPP IV substrates Tetrapeptide Prodrugs (IC50) pH 2 pH 8ChgProValboroPro  65 nM  72 nM ChgProAlaboroPro  19 nM  15 nMChgAlaValboroPro 190 nM 360 nM ChgProChgboroAla 180 nM 170 nMAlaProValboroPro  20 nM  21 nM GlyProValboroPro  17 nM  46 nMProProProboroPro  10 μM N/A** GlyProProboroPro  9 μM  20 mMAlaProProboroPro  8 mM N/A AlaAlaProboroPro  3 mM  2 mM ChgAlaProboroPro 2 mM  30 mM

TABLE 2 Toxicity of Val-boroPro in rats and mice. Results show thatVal-boroPro is quite toxic to rats but not to mice. TEST DOSE NO. OFANIMAL PROJECT # ARTICLE GROUP LEVEL ROUTE SPECIES DOSED MORTALITY002898 Val-ProBoronic 1    5 mg/kg Oral Zucker 4 4 acid Obese OralZucker Lean 3 3 002969 Val-ProBoronic 2 0.035 mg/kg Oral Zucker 4 4 acidObese Oral Zucker Lean 4 1 3  0.35 mg/kg Oral Zucker 4 4 Obese OralZucker Lean 4 4 4  1.75 mg/kg Oral Zucker 4 2 Obese Oral Zucker Lean 4 2010757 Val-ProBoronic 3 0.035 mg/kg Diabetic 7 0 acid Mice 4  0.35 mg/kgDiabetic 7 0 Mice 5  3.5 mg/kg Diabetic 7 0 Mice 011050 Val-ProBoronic 20.035 mg/kg Diabetic 8 0 acid (starved) Mice 3 0.035 mg/kg Diabetic 8 0Mice

TABLE 3 In vivo inhibition of serum DPP IV as a function of timefollowing three different doses levels of CHG-Pro-Ala-boroPro,Val-boroPro, and CHG-Pro-Val- boroPro. Normal- Diabetic- control controlCHGProAlaboroPro ValboroPro CHGProValboroPro 0.025 mg/kg 1 hr Post 82.65100.00 111.42 47.49 34.70 Rx- Glucose 4 hrs 89.95 100.00 58.85 39.2324.40 Post Rx- Glucose 0.01 mg/kg 1 hr 82.65 100.00 100.00 59.36 48.86Post Rx- Glucose 4 hrs 89.95 100.00 77.51 58.85 40.67 Post Rx- Glucose0.0025 mg/kg 1 hr 82.65 100.00 109.59 94.52 84.47 Post Rx- Glucose 4 hrs89.95 100.00 99.52 93.30 81.82 Post Rx- Glucose

TABLE 4 IC₅₀ Values at Ten Minutes for DPPIV Prodrugs pH 2 pH 8 pH 2 pH8 P₃ Proline Analogues ChgAzeEtgboroPro 15 nM 16 nM ProboroPro ProdrugsProProProboroPro 10 μM N/A ChgDhpEtgboroPro 15 nM 17 nM AlaProProboroPro8.3 μM N/A ChgHypEtgboroPro 17 nM 25 nM GlyProProboroPro 8.6 μM 20 μMChgPipEtgboroPro 19 nM 14 nM AlaAlaProboroPro 3 μM N/A ChgThz4EtgboroPro13 nM 16 nM ChgAlaProboroPro 2 μM 30 μM β-Casomorphin TyrProAlaboroPro15 nM 17 nM boroAla Prodrugs ChgProChgboroAla 180 nM 170 nM AnaloguesPheProAlaboroPro 6 nM 8 nM ChgAlaEtgboroAla 1.3 μM 2.5 μMTyrProPheboroPro 50 nM 60 nM ChgProtBugboroAla 6.8 μM 8.4 μM P₂ BranchedProdrugs ChgProAlaboroPro 17 nM 17 nM ValboroPro ProdrugsAlaProValboroPro 20 nM 21 nM ChgProEtgboroPro 12 nM 14 nMGlyProValboroPro 17 nM 46 nM ChgProValboroPro 70 nM 70 nMChgAlaValboroPro 190 nM 360 nM ChgProBugboroPro 1.5 μM 8 μMAlaAlaValboroPro 2 μM 25 μM AibAlaValboroPro 2 μM N/A N-Methyl ProdrugsN-Me-PheProAlaboroPro 2 μM 2.7 μM Miscellaneous ChgAlaEtgboroPro 600 nM802 nM SarDhpAlaboroPro 39 nM 34 nM ChgProChgboroPro 14 nM 14 nMtBugProAlaboroPro 14 nM 18 nM

TABLE 5 Study Design for Evaluation of dpp iv inhibition followingsingle oral administration of new chemical entities to male spraguedawley rats Test Target Dose Group Article Route of Dose Volume No.Samples ID ID Administration (mg/kg) (mL/kg) Rats Rat ID Collected 1Oral 0.05 2 4 1001-1004 Blood, Plasma 2 0.5 4 2001-2004 Blood, Plasma 35 4 3001-3004 Blood, Plasma 4 0.05 4 4001-4004 Blood, Plasma 5 0.5 45001-5004 Blood, Plasma 6 5 4 6001-6004 Blood, Plasma 7 0.05 4 7001-7004Blood, Plasma 8 0.5 4 8001-8004 Blood, Plasma 9 5 4 9001-9004 Blood,Plasma

TABLE 6 Study Design for evaluation of DPP IV inhibition followingsingle oral administration of four triad compounds in male rats DoseLevel Group No. of Test (mg ALA- Samples ID Rats Route Article PRO/kg*)Rat ID Collected 1 3 Oral 1 0.5 1001-1003 Blood, Plasma 2 3 Oral 1 2.52001-2003 Blood, Plasma 3 3 Oral 1 10.0 3001-3003 Blood, Plasma 4 3 Oral2 0.5 4001-4003 Blood, Plasma 5 3 Oral 2 2.5 5001-5003 Blood, Plasma 6 3Oral 2 10.0 6001-6003 Blood, Plasma 7 3 Oral 3 0.5 7001-7003 Blood,Plasma 8 3 Oral 3 2.5 8001-8003 Blood, Plasma 9 3 Oral 3 10.0 9001-9003Blood, Plasma *Expressed as mg equivalent; applies to Test Articles 1, 2and 3 only Test Article 1: Chg-PRO-ALA-PRO Boronic acid (administered asALA-PRO freebase) Test Article 2: N-ME-PHE-PRO-ALA-PRO Boronic acid(administered as ALA-PRO freebase) Test Article 3: N-ME-GLY-PRO-ALA-PROBoronic acid (administered as ALA-PRO freebase)

TABLE 7 Summary of Clinical Signs after DPP IV inhibition followingsingle oral administration of four triad compounds in male ratsApproximate Approximate Time of Sacrifice Onset of or Time AnimalObservation was Found Animal (Time Dead ID post-dose) ClinicalObservation (hr post-dose) L-2-Chg-PRO-ALA-PRO boronic acid 0.5 mg/kg*1001 — None — 1002 — None — 1003 — None — L-2-Chg-PRO-ALA-PRO boronicacid 2.5 mg/kg* 2001 — None — 2002 — None — 2003 — None —L-2-Chg-PRO-ALA-PRO boronic acid 10.0 mg/kg* 3001 2 hr Redness of earsand paws 5 hr (sacrificed) 4 to 5 hr Decreased activity 5 hr laboredbreathing 3002 — None — 3003 — None — N-Me-Phe-PRO-ALA-PRO boronic acid0.5 mg/kg* 4001 — None — 4002 — None — 4003 — None —N-Me-Phe-PRO-ALA-PRO boronic acid 2.5 mg/kg* 5001 2 hr Redness of earsand paws — 5002 — None — 5003 — None — N-Me-Phe-PRO-ALA-PRO boronic acid10.0 mg/kg* 6001 — None — 6002 4 hr Redness of ears and paws — 6003 —None — N-Me-Gly-3,4 dehydroproline-ALA-PRO boronic acid 0.5 mg/kg* 7001— None — 7002 8 hr Redness of ears and paws — 7003 — None — N-Me-Gly-3,4dehydroproline-ALA-PRO boronic acid 2.5 mg/kg* 8001 2 hr Redness of earsand paws 4 hr (sacrificed) 4 hrr Decreased activity, soft feces, laboredbreathing, cold to touch 8002 4 hr Redness of ears and paws — 8003 —None — N-Me-Gly-3,4 dehydroproline-ALA-PRO boronic acid 10.0 mg/kg* 9001— None 7 hr (found dead) 9002 — None — 9003 — None — *Corresponds toALA-PRO boronic dose equivalents

1-28. (canceled)
 29. A prodrug represented by formula (I) or a solvate,pharmaceutically functional derivative or pharmaceutically acceptablesalt thereof:A-G  (I) wherein A represents a peptidyl moiety, 2-10 amino acidresidues in length, that is a substrate for an activating protease whichis associated with tumors; A and G are covalently linked by a bond thatis cleaved by the activating protease; and G represents a dipeptidylproteosome inhibitor moiety that, when released from the prodrug bycleavage by the activating protease, inhibits the proteolytic activityof a proteosome with a Ki of 100 nM or less, wherein, the dipeptidylproteosome inhibitor moiety G, when cleaved from A by the activatingprotease, undergoes reversible cyclization-dependent inactivation overtime.
 30. The prodrug of claim 29, wherein A is a dipeptidyl ortripeptidyl moiety.
 31. The prodrug of claim 29, wherein at least oneresidue of A is a non-naturally occurring amino acid analog.
 32. Theprodrug of claim 29, wherein at least one residue of A is a D-aminoacid.
 33. The prodrug of claim 29, wherein the amino terminus of A isblocked with an amino-terminal protecting group.
 34. The prodrug ofclaim 33, wherein the amino-terminal protecting group is an acyl group.35. The prodrug of claim 34, wherein the acyl group is selected from thegroup consisting of formyl, dansyl, acetyl, benzoyl, trifluoroacetyl,succinyl and methoxysuccinyl.
 36. The prodrug of claim 29, wherein thedipeptidyl proteosome inhibitor moiety G is represented by formula (II):Xaa₁-Xaa₂-W  (II) wherein Xaa₁ and Xaa₂ each independently represent anamino acid residue; W represents —CN, —CH═NR₅,

R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,—(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH, —(CH₂)n-S-alkyl,—(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl, —(CH₂)n-S—(CH₂)_(m)—R₆,—C(O)C(O)NH₂, —C(O)C(O)OR₇; R₆ represents, independently for eachoccurrence, a substituted or unsubstituted aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; R₇ represents, independently for eachoccurrence, hydrogen, or a substituted or unsubstituted alkyl, alkenyl,aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; Y₁ and Y₂ canindependently or together be OH, or a group capable of being hydrolyzedto a hydroxyl group, including cyclic derivatives where Y₁ and Y₂ areconnected via a ring having from 5 to 8 atoms in the ring structure; R₅₀represents O or S; R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇; R₅₂represents hydrogen, a lower alkyl, an amine, —OR₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure; X₁ represents a halogen;X₂ and X₃ each represent a hydrogen or a halogen; m is zero or aninteger in the range of 1 to 8; and n is an integer in the range of 1 to8.
 37. The prodrug of claim 36, wherein W is

and Y₁ and Y₂ are OH.
 38. The prodrug of claim 36, wherein Xaa₁ or Xaa₂is a non-naturally occurring amino acid analog.
 39. The prodrug of claim29, wherein the activating protease is Fibroblast Activating Protein(FAP), Prostate Specific Antigen (PSA), Dipeptidylpeptidase IV (DPIV),matriptase or falcipain.
 40. The prodrug of claim 39, wherein theactivating protease is Fibroblast Activating Protein (FAP).
 41. Theprodrug of claim 29, wherein the reversible cyclization-dependentinactivation comprises the dipeptidyl proteosome inhibitor moiety Gundergoing inter-conversion between linear and cyclic forms, wherein thecyclic form has a Ki for inhibiting the proteosome at least 2 timesgreater than the linear form.
 42. The prodrug claim 29, wherein thecyclic form has a Ki at least 10 times the Ki of the linear form. 43.The prodrug of claim 29, wherein the cyclic form has a Ki at least 100times the Ki of the linear form.
 44. The prodrug of claim 29, whereinthe prodrug has a therapeutic index at least two times greater than thetherapeutic index for the dipeptidyl proteosome inhibitor moiety G whenadministered alone.
 45. A pharmaceutical composition, comprising apharmaceutically acceptable carrier; and a prodrug of claim 29, or apharmaceutically acceptable salt thereof.
 46. A packaged pharmaceutical,comprising one or more prodrugs of claim 29 formulated in apharmaceutically acceptable excipient, in association with instructions(written and/or pictorial) describing the recommended dosage and/oradministration of the formulation to a patient.